Pharmaceutical preparation of carbohydrates for therapeutic use

ABSTRACT

The disclosure provides methods for preparation of carbohydrate replacement therapies (CRT) that include nanocarriers of carbohydrates and glycolipids for pharmaceutical delivery to cell interior, endoplasmic reticulum, and Golgi for treating CDG type I and CDG type II diseases as well as other metabolic disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/878,591, filed Sep. 16, 2013, which is hereby incorporated byreference in its entirety.

FIELD

The present invention relates to compositions containing carbohydratesencapsulated in a lipid particle for delivery to the cell interior,methods of delivering the encapsulated carbohydrates into the interiorof cells, and methods of using such compositions for treating diseasesand disorders, such as congenital disorders of glycosylation (CDG). Forexample, the present invention relates to methods for preparation ofcarbohydrates and glycolipids for pharmaceutical delivery to cellinterior, endoplasmic reticulum (ER), and Golgi.

BACKGROUND

Glycosylation, the enzymatic attachment of carbohydrates (glycans) toproteins and lipids is a co-translational and post-translationalmodification (PTM) that is more common than any other PTM as it appliesto a majority of proteins synthesized in the rough endoplasmic reticulum(ER). Glycosylation plays a critical role in a variety of biologicalprocesses of membrane and secreted proteins. In the ER, glycosylationdefines protein structure and folding and acts as a quality controlmechanism that dictates the export of properly folded proteins to Golgior targets misfolded ones for degradation. Glycan moieties may also actas ligands for cell surface receptors to mediate cell attachment orstimulate signal transduction pathways. Congenital disorders ofglycosylation, also known as CDG syndromes, are a group of rare geneticdiseases where tissue proteins and/or lipids carry defectiveglycosylation and/or lack of glycosylation. These diseases are linked tonumerous enzymatic deficiencies and often times cause severe, sometimesfatal, impairments of the nervous system, muscles, intestines, andseveral other organ systems.

Common clinical symptoms in children with CDG include hypotonia,developmental delay, failure to thrive, hepatic dysfunction,coagulopathy, hypothyroidism, esotropia, abnormal fat pattern andinverted nipples, hypoglycemia, seizure, cerebellar hypoplasia, andstroke-like episodes in a developmentally delayed child. At an olderage, in adolescence, and adulthood, the presentation may include ataxia,cognitive impairment, and absence of puberty in females, small testes inmales, retinitis pigmentosa, scoliosis, joint contractures, andperipheral neuropathy.

CDG may be classified into two groups: CDG type I and CDG type II. CDGtype I is characterized by defects in the initial steps of N-linkedprotein glycosylation, i.e., biosynthesis of dolichol pyrophosphatelinked oligosaccharide (DLO), which occur in the ER, or transfer of theDLO to asparagine residues of nascent polypeptides. CDG type II involvesdefects in further processing (synthetic or hydrolytic) of theprotein-bound glycan. Currently, twenty-two CDG type I and fourteen typeII variants have been identified. One of the most common subtype of CDGis CDG-Ia (approximately 70% of all CDG cases), which is characterizedby loss or reduction of phosphomannomutase 2 (PMM) activity leading todeficiency or insufficiency in intracellular N-glycosylation (Jaeken etal. J. of Inherit. Met. Disease, 2008, 31:669-672). PMM is responsiblefor the conversion of mannose-6-phosphate to mannose-1-phosphate.

Although several different approaches to developing therapies for CDGhave been explored, researchers continue their search for a suitablecure or a therapy for mitigating the disease itself. Existing treatmentsfor manifestations include, for example, nutritional supplements, tubefeeding, and a wide range of therapies that attempt to treatgastro-esophageal reflux, persistent vomiting, developmental delays,ocular abnormalities, and hypothyroidism. Patients also requireintravenous (IV) hydration and physical therapy for stroke-likeepisodes. Adults with orthopedic Symptoms often require wheel chairs,transfer devices, and surgical treatment for scoliosis (Sparks et al.,Disorders of Glycosylation Overview. 2005 In: Pagon R A, Adam M P, BirdT D, et al., editors, GeneReviews™, Seattle (Wash.): University ofWashington, Seattle; 1993-2013).

Currently, CDG-Ib is the only known CDG for which a relatively effectivetreatment is available, namely oral D-mannose administration. However,such therapy may not be as effective in treating CDG-Ia patients andthere are currently limited treatment options for other CDG type Isubtypes and CDG type II diseases. One of the reasons for a lack inestablished therapy for CDG-I disorders may be due to the plethora ofheterogeneous clinical phenotypes presented that do not show a directcorrelation to the PMM enzyme activity.

Patients suffering from a reduction in PMM activity have reducedproduction of mannose-1-phosphate (Man-1-P), associated with symptoms ofmultivisceral impairments. In order to overcome PMM productiondeficiency, it is important to supply downstream enzymes with therequired substrate (i.e., Man-1-P). However, the delivery andmaintenance of such a systemic supply of Man-1-P is problematic, asextracellular enzymes within bodily fluids degrade Man-1-P whendelivered exogenously by oral or intravenous administration. Anotherproblem with exogenously delivered Man-1-P is that its high polarityprevents Man-1-P from penetrating into the cell interior (i.e., cytosol)and thus treating the deficiency in PMM production.

Derivatives of the polar Man-1-P can be synthesized to make Man-1-P morecell-permeable (US Patent Publication No. 2009/0054353). This approach,however, is also problematic, as the cell-permeable Man-1-P derivativehas been shown to be either unstable for clinical use or cytotoxic viathe by-products of the Man-1-P derivative (Eklund et al., Glycobiology2005, 15: 1084-1093; Rutschow et al. Bioorg Med Chem 2002, 10:4043-4049; and Hardre et al., Bioorg Med Chem Lett 2007, 17: 152-155).

Other potential therapies have focused on inhibiting enzymes thatcatabolize mannose-6-phosphate (Man-6-P), a precursor to Man-1-P, viainhibition of phosphomannose isomerases (PMI). The approach focuses onforcing the reaction towards optimizing homeostasis, which with the useof PMI inhibitors, would have been skewed toward production of Man-6-P.These approaches, however, are ineffective as clinical treatment optionsdue to their associated toxicity, off-target side effects, and poorselective tissue penetration.

Accordingly, unmet needs exist for improved compositions and methods fordelivering carbohydrates, such as Man-1-P, to the cell interior in orderto treat disorders, such as a congenital disorder of glycosylation(CDG), to subjects (including, for example, humans) in need of suchtreatment.

BRIEF SUMMARY

The present disclosure meets the unmet needs described above byproviding carbohydrates encapsulated by nanocarriers that address thecytotoxicity and stability problems associated with eitherpermeabilizing cells or using carbohydrate derivatives. Suchcarbohydrates may be endogenous carbohydrates, including, for example,mannose-1-phosphate. By utilizing endocytotic pathways, nanocarriers canenter the cell interior and deliver the carbohydrate into the cytosol ofa cell. This implementation of nanocarriers encapsulating carbohydrates,facilitates simultaneously the ease of therapy, allowing for higherdosages of the carbohydrate to reach the biochemical glycosylationpathway, as well as potential easement on administration. In someaspects, the disclosure provides methods for preparation ofcarbohydrates and glycolipids using nanocarriers for pharmaceuticaldelivery to cell interior, endoplasmic reticulum, and Golgi. Thedisclosure provides carbohydrate replacement therapies for, but notlimited to, treating diseases of CDG type I and II.

The present disclosure also addresses unmet needs related to effectivetherapeutics for diseases, such as diseases of CDG, as well as issuesrelated to the delivery of such therapeutics. In some aspects, thedisclosure provides a method of delivering carbohydrates such asmannose-1-phosphate to the cytosol of cells. Without wishing to be boundby any theory, it is believed that such method of deliver thecarbohydrates (e.g., as part of a medicament) would bypass the work ofgenetically defective cytosolic enzymes, namely phosphomannomutase (PMM)and phosphomannose isomerase (PMI), which are found prevalent in CDG Idisorders.

In some embodiments, the compositions of the present disclosure mayinvolve the delivery of, for example, mannose-1-phosphate into thecytosol of the cell using nanocarriers. In particular, biodegradable andbiocompatible lipid-sterol-water lamellar nanostructures may be used toencapsulate the carbohydrate. The disclosed medicament represents themost potent and viable treatment option for disorders, such as the CDGIa disorder.

In certain embodiments, the present disclosure provides compositionscontaining a lipid particle and carbohydrates, such as endogenouscarbohydrates, encapsulated in the lipid particle, and methods of usingsuch compositions to deliver the carbohydrate to the interior of cellsand to treat diseases and disorders, such as congenital disorders ofglycosylation (CDG).

In some embodiments, the compositions described herein are medicaments.Such compositions (e.g., medicaments) may include an aqueous suspensionof liposomes encasing, for example, mannose-1-phosphate. Liposomes maybe prepared from vesicle-forming lipids, such as but not limited tophosphatidylcholine (PC). The liposome may also be prepared inconjunction with a sterol, namely cholesterol. The full liposome may beenriched with polyethylene-glycol (PEG), or PEGylated, in order toenhance circulation and retention with the body fluids uponadministration, as well as protect the liposome from prematuredegradation and excretion of the medicament from the body of a subjectin need of such treatment.

Accordingly, certain aspects of the present disclosure relate to acomposition comprising a lipid particle; and an endogenous carbohydrateencapsulated in the lipid particle, where the lipid particle contains amolecule that is capable of minimizing degradation of the lipid particleand/or enhancing retention of the lipid particle when administered to asubject, and/or makes the lipid particle immunotolerant whenadministered to a subject. In some embodiments, the molecule is astealth molecule, such as ethylene oxide, an ethylene oxide oligomer, anethylene oxide polymer, polyethylene glycol (PEG), or any combinationthereof; and/or a PEGylated neutral lipid.

Other aspects of the present disclosure relate to a compositioncontaining a lipid particle that contains ethylene oxide, an ethyleneoxide oligomer, an ethylene oxide polymer, polyethylene glycol (PEG), orany combination thereof; and a carbohydrate encapsulated in the lipidparticle. Other aspects of the present disclosure relate to acomposition containing a lipid particle; and a carbohydrate encapsulatedin the lipid particle, wherein the lipid particle contains choline,ethanolamine, glycerol, inositol, or any combination thereof. In someembodiments, the lipid particle further includes a molecule, such as astealth molecule, that is capable of minimizing degradation of the lipidparticle and/or enhancing retention of the lipid particle whenadministered to a subject, and/or makes the lipid particleimmunotolerant when administered to a subject. In some embodiments, thelipid particle is selected from a liposome, a micelle, a solid lipidnanoparticle, and a niosome. In some embodiments, the lipid particle isa liposome. In some embodiments, the liposome is a stealth liposome thatmay be immunotolerant. In some embodiments, the endogenous carbohydrateand/or carbohydrate is selected from a monosaccharide, a phosphorylatedmonosaccharide, a disaccharide, a phosphorylated disaccharide, anoligosaccharide, a phosphorylated oligosaccharide, a polysaccharide, aphosphorylated polysaccharide, mannose, a phosphorylated mannose, amannofuranose, a phosphorylated mannofuranose, a mannopyranos, aphosphorylated mannopyranos, mannose-1-phosphate, a nucleotide sugar, auridine diphosphate, a guanine diphosphate, a cytosine monophosphate,fucose, GDP-fucose, a sialic acid, CMP-sialic acid, N-acetylneuraminicacid (Neu5Ac), CMP-Neu5Ac, and derivatives thereof. In some embodiments,the endogenous carbohydrate is mannose-1-phosphate.

Other aspects of the present disclosure relate to a compositioncontaining a liposome; and mannose-1-phosphate encapsulated in theliposome, wherein the liposome comprises cholesterol andphosphatidylethanolamine (PE) attached to polyethylene glycol (PEG).

Other aspects of the present disclosure relate to a pharmaceuticalcomposition containing a composition of any of the precedingembodiments, and a pharmaceutically acceptable carrier.

Other aspects of the present disclosure relate to a kit containing acomposition of any of the preceding embodiments for use in any of themethods described herein.

Other aspects of the present disclosure relate to a method fordelivering a carbohydrate to a subject in need thereof, by administeringto the subject a composition of any of the preceding embodiments. Insome embodiments, the composition is administered orally, topically,dermally, nasally, intravenously, intramuscularly, intraperitoneally,intracerobrospinally, intracranially, intraspinally, subcutaneously,intraarticularly, intrasynovialy, or intrathecaly.

Other aspects of the present disclosure relate to a method fordelivering a carbohydrate to a cell interior of a subject in needthereof, by administering to the subject a composition of any of thepreceding embodiments. In some embodiments, the composition isadministered orally, topically, dermally, nasally, intravenously,intramuscularly, intraperitoneally, intracerobrospinally,intracranially, intraspinally, subcutaneously, intra-articularly,intrasynovialy, or intrathecaly.

Other aspects of the present disclosure relate to a method for treatinga congenital disorder of glycosylation (CDG) in a subject in needthereof, by administering to the subject a composition of any of thepreceding embodiments. In some embodiments, the congenital disorder ofglycosylation (CDG) is selected from a CDG type I disorder, a CDG-Iadisorder, a CDG type II disorder, a CDG-IIc disorder, and a CDG-IIfdisorder. In some embodiments, the congenital disorder of glycosylation(CDG) is a CDG-Ia disorder. In some embodiments, the composition isadministered orally, topically, dermally, nasally, intravenously,intramuscularly, intraperitoneally, intracerobrospinally,intracranially, intraspinally, subcutaneously, intra-articularly,intrasynovialy, or intrathecaly.

DESCRIPTION OF THE DRAWINGS

The following enumerated embodiments are representative of some aspectsof the invention.

FIG. 1A depicts an exemplary structure of mannose-1-phosphate (M1P).FIG. 1B depicts an exemplary structure of polyethylene glycol (PEG).FIG. 1C depicts an exemplary structure of phosphatidylethanolamine (PE)attached to PEG. FIG. 1D depicts an exemplary structure ofphosphatidylcholine (PC).

FIG. 2 depicts an exemplary the structure of a liposome compositioncontaining a carbohydrate encapsulated within the core of the liposome.

DETAILED DESCRIPTION

Provided herein are pharmaceutical nanocarriers of carbohydrates andglycolipids (e.g., lipid compositions containing a lipid particle andone or more carbohydrates) that are capable of delivering thecarbohydrates and/or glycolipids to a cell interior including, withoutlimitation, the cytoplasm, endoplasmic reticulum, and Golgi. Suitablenanocarriers include, but are not limited to, liposomes, micelles, solidlipid nanoparticles, and niosomes.

As disclosed herein, the composition of suitable nanocarriers is notlimited to any class and molecular weight of lipids, polyethyleneglycol, or their derivatives. In some embodiments, the presentdisclosure relates more specifically, but is not limited to, liposomalpreparations of mannose-1-phosphate, guanosine 5′-diphospho-beta-L-fucose (GDP-fucose), cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-sialic acid), and dolicholpyrophosphate linked oligosaccharide, Glc3Man9GlcNAc2-PP-Dol, whereGlc=glucose, Man=mannose, GlcNAc=N-Acetylglucosamine, P=phosphate,Dol=dolichol. In some variations, the dolicholmay have chain lengths ofat least 10 isoprene units, and in certain variations, 14-18 isopreneunits.

As disclosed herein in some embodiments, carbohydrates and glycolipidsof the present disclosure can be integrated into liposomes designed forspecific targeting to cell interior, ER, or Golgi. A chemoenzymaticapproach to synthesis of dolichol pyrophosphate linked oligosaccharidemay be utilized (Wang Z. et al., Science, 2013, 341, 379-383 andWeerapana E. et al., J. Am. Chem. Soc., 2005, 127, 13766-13767). In someembodiments, liposomal preparations may be optimized for each targetedcell compartment as described in Torchilin V. et al., Curr. ProteinPept. Sci., 2003, 4, 133-140; Pollock S. et al., FASEB, 2010, 24,1866-1878; Fujiwara T. et al., Int. J. Pharm., 2010, 386, 122-130; andWO 2009/118658 A2.

In other embodiments, architectural modification to a liposome mayinclude, but not limited to, cholesteryl hemisuccinate (CH) andpolyethylene glycol (PEG) chains. As disclosed herein, suchmodifications can shield carbohydrate and glycolipid moieties of thecustomized nanoparticle from degradation and will enhance circulationand/or retention time of the pharmaceutical within the body.

Accordingly, the present disclosure provides lipid compositionscontaining a lipid particle and one or more carbohydrates, such asendogenous carbohydrates, encapsulated in the lipid particle, where thelipid compositions are capable of delivering the carbohydrate into theinterior of a cell. In some embodiments, such carbohydrate-carryinglipid compositions are useful for treating, preventing, or reducing riskof a congenital disorder of glycosylation (CDG) in a subject in needthereof. In some embodiments, the lipid particle may be a liposome, amicelle, a solid lipid nanoparticle, or a noisome. In some embodiments,the one or more carbohydrates may be mannose-1-phosphate, guanosine5′-diphospho-beta-L-fucose (GDP-fucose), and/or cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-sialic acid). In someembodiments, the lipid particle contains a molecule that is capable ofminimizing degradation of the lipid particle and/or enhancing retentionof the lipid particle when administered to a subject, and/or makes thelipid particle immunotolerant when administered to a subject. Asdisclosed herein, such molecules may shield, or otherwise protect, thelipid particle and encapsulated carbohydrate of the present disclosurefrom degradation, and may also enhance cell permeability, circulationand/or retention time of the lipid particle when the lipid particle isadministered to a subject in need thereof. Additionally, such moleculescan allow the lipid particle to avoid detection by the immune system ofa subject in need thereof that is administered the lipid particle, andas such are considered stealth molecules. Accordingly, in someembodiments, the molecule is a stealth molecule that is capable ofinducing immunotolerance when administered to a subject in need thereof.Examples of such suitable molecules include, without limitation,ethylene oxide, an ethylene oxide oligomer, an ethylene oxide polymer,polyethylene glycol (PEG), or any combination thereof; and/or aPEGylated neutral lipid.

Thus, in certain aspects, the present disclosure provides a compositioncontaining a lipid particle; and an endogenous carbohydrate encapsulatedin the lipid particle, where the lipid particle contains a molecule thatis capable of minimizing degradation of the lipid particle and/orenhancing retention of the lipid particle when administered to asubject, and/or makes the lipid particle immunotolerant whenadministered to a subject. In some embodiments the molecule is a stealthmolecule, such as ethylene oxide, an ethylene oxide oligomer, anethylene oxide polymer, polyethylene glycol (PEG), or any combinationthereof; and/or a PEGylated neutral lipid.

In certain aspects, the present disclosure provides a compositioncontaining a lipid particle comprising ethylene oxide, an ethylene oxideoligomer, an ethylene oxide polymer, polyethylene glycol (PEG), or anycombination thereof; and a carbohydrate encapsulated in the lipidparticle.

In certain aspects, the present disclosure provides a compositioncontaining a lipid particle; and a carbohydrate encapsulated in thelipid particle, wherein the lipid particle comprises choline,ethanolamine, glycerol, inositol, or any combination thereof. In someembodiments, the lipid particle contains a molecule that is capable ofminimizing degradation of the lipid particle and/or enhancing retentionof the lipid particle when administered to a subject, and/or makes thelipid particle immunotolerant when administered to a subject. In someembodiments the molecule is a stealth molecule, such as ethylene oxide,an ethylene oxide oligomer, an ethylene oxide polymer, polyethyleneglycol (PEG), or any combination thereof; and/or a PEGylated neutrallipid.

In certain aspects, the present disclosure provides a compositioncontaining a liposome; and mannose-1-phosphate encapsulated in theliposome, wherein the liposome comprises cholesterol andphosphatidylethanolamine (PE) attached to polyethylene glycol (PEG).

Additionally, pharmaceutical compositions are provided containing acarbohydrate-containing lipid composition of the present disclosure incombination with pharmaceutically acceptable carriers. The presentdisclosure also provides a kit containing a carbohydrate-containinglipid composition of the present disclosure for use in any of themethods described herein.

The present disclosure further provides methods of using acarbohydrate-containing lipid composition of the present disclosure todeliver a carbohydrate to a subject in need thereof. The presentdisclosure further provides methods of using a carbohydrate-containinglipid composition of the present disclosure to deliver a carbohydrate toa cell interior to a subject in need thereof. The present disclosurefurther provides methods of using a carbohydrate-containing lipidcomposition of the present disclosure to treat, prevent, or reduce riskof a congenital disorder of glycosylation (CDG) in a subject in needthereof.

Lipid Particles

Certain aspects of the present disclosure relate to compositionscontaining lipid particles that contain carbohydrates encapsulatedwithin the lipid particle.

As used herein, the term “lipid particle” refers to particles formed bylipids in an aqueous solution. Suitable examples of lipid particlesinclude, without limitation, liposomes, micelles, solid lipidnanoparticles, niosome, lipospheres, emulsomes, and emulsions.

As used herein, a carbohydrate that is “encapsulated” in a lipidparticle, refers to a lipid particle that provides an active agent ortherapeutic agent, such as a carbohydrate of the present disclosure,with full encapsulation, partial encapsulation, or both. In somevariations, at least a portion of the carbohydrate may be “encapsulated”by a lipid particle and localized within the core of a lipid particleand/or within the inner surface (e.g., the membrane) of a lipidparticle. Alternatively, in other variations, the entire carbohydratemay be “encapsulated” by a lipid particle and localized within the coreof a lipid particle and/or within the inner surface (e.g., the membrane)of a lipid particle.

Any lipid particle known in the art suitable for delivering anencapsulated carbohydrate of the present disclosure to the interior of acell may be used. Examples of suitable lipid particles include, withoutlimitation, liposomes, micelles, solid lipid nanoparticles, andniosomes.

In some embodiments, lipid particles of the present disclosure have anaverage particle size that ranges from about 0.02 microns in diameter toabout 0.5 microns in diameter. In certain embodiments, lipid particlesof the present disclosure have an average particle size of about 0.02microns in diameter, about 0.03 microns in diameter, about 0.04 micronsin diameter, about 0.05 microns in diameter, about 0.06 microns indiameter, about 0.07 microns in diameter, about 0.08 microns indiameter, about 0.09 microns in diameter, about 0.10 microns indiameter, about 0.15 microns in diameter, about 0.20 microns indiameter, about 0.25 microns in diameter, about 0.30 microns indiameter, about 0.35 microns in diameter, about 0.40 microns indiameter, about 0.45 microns in diameter, or about 0.50 microns indiameter.

In some embodiments, lipid particles of the present disclosure arecapable of delivering a carbohydrate of the present disclosure to theinterior of a cell, such as the cytoplasm, endoplasmic reticulum, orGolgi.

In some embodiments, lipid particles of the present disclosure may beoptimized for targeting to particular organelles within a cell,including without limitation, the endoplasmic reticulum (ER), Golgi, andlysosome. In some embodiments, lipid particles of the present disclosuremay be optimized for targeting to particular tissues and/or organs in asubject. Methods of optimizing lipid particles for targeting specificorganelles, tissues, and organs are well known in the art (e.g.,Torchilin V. et al., Curr. Protein Pept. Sci., 2003, 4, 133-140;Torchilin V. et al., J. Drug Target., 2011, 19, 606-614; Huwyler J. etal., J. Pharmacol. Exp. Ther., 1997, 282: 1541-1546; Pollock S. et al.,FASEB, 2010, 24, 1866-1878; Fujiwara T. et al., Int. J. Pharm., 2010,386, 122-130; and WO 2009/118658).

In some embodiments, lipid particles of the present disclosure contain acarbohydrate of the present disclosure, that when delivered to theinterior of a cell of a subject (including, for example, a human) inneed thereof can induce at least a 0.05-fold to at least a 10-foldincrease in cellular production of higher-order lipid-linkedoligosaccharides, as compared to cellular production of higher-orderlipid-linked oligosaccharides in the absence of lipid particles of thepresent disclosure containing a carbohydrate of the present disclosure.In some embodiments, the lipid particles provided herein, whenadministered to a subject (including, for example, a human) in needthereof, may induce at least a 0.05-fold, at least a 0.1-fold, at leasta 0.2-fold, at least a 0.3-fold, at least a 0.4-fold, at least a0.5-fold, at least a 0.6-fold, at least a 0.7-fold, at least a 0.8-fold,at least a 0.9-fold, at least a 1-fold, at least a 1.5-fold, at least a2-fold, at least a 2.5-fold, at least a 3-fold, at least a 3.5-fold, atleast a 4-fold, at least a 4.5-fold, at least a 5-fold, at least a5.5-fold, at least a 6-fold, at least a 6.5-fold, at least a 7-fold, atleast a 7.5-fold, at least an 8-fold, at least an 8.5-fold, at least a9-fold, at least a 9.5-fold, or at least a 10-fold increase in cellularproduction of higher-order lipid-linked oligosaccharides in the subject,as compared to cellular production of higher-order lipid-linkedoligosaccharides in the subject in the absence of administering suchlipid particles to the subject.

As used herein a “higher-order lipid-linked oligosaccharide” refers toan oligosaccharide having at least five monosaccharide subunits and thatis linked to a lipid. For example, a higher-order linked oligosaccharidemay refer to an oligosaccharide containing at least four mannosesubunits (Man4), five mannose subunits (Man5), six mannose subunits(Man6), seven mannose subunits (Man7), eight mannose subunits (Man8), ornine mannose subunits (Man9) and an N-acetylglucosamine disaccharide(GlcNAc2), where the oligosaccharide is linked to a dolichol. In someembodiments, the higher-order lipid linked oligosaccharides may includean oligosaccharide portion that includes Man4GlcNAc2, Man5GlcNAc2,Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man8GlcNAc2, or any combinationthereof.

Liposomes

In some embodiments, a lipid particle of the present disclosure may be aliposome. As used herein, a “liposome” refers to a vesicle composed of alamellar phase lipid bilayer. Any suitable liposome known in the art maybe used. In some embodiments, the liposome has a lamellar nanostructure.As used herein, the term “lamellar nanostructure” refers to ananostructure, such as a lipid particle, that includes parallelamphiphilic bilayers separated by a lumen.

Liposomes of the present disclosure may be prepared by any suitablemethod known in the art and disclosed herein. Examples of suitablemethods for preparing liposomes include, without limitation, disruptingbiological membranes, such as by mechanical dispersion includingsonication, thin-film hydration, emulsions, french pressure cell,extrusion, and reconstitution of dried vesicles; solvent dispersionincluding ethanol injection, ether injection, double emulsion, reversephase, and vaporization; and detergent removal methods.

In certain embodiments, the liposome is a stealth liposome that may beimmunotolerant. As used herein, the term “stealth liposome” refers toliposomes that are capable of avoiding detection by a subject's immunesystem. As such, a stealth liposome may be immunotolerant. For example,the subject may be a human.

Micelles

In some embodiments, a lipid particle of the present disclosure may be amicelle. As used herein, a “micelle” refers to an aggregate ofsurfactant molecules (e.g., soaps, detergents, fatty acids, lipids,phospholipids, etc.) that are dispersed in a liquid colloid. A micellein aqueous solution may form an aggregate with hydrophilic head regionsin contact with surrounding solvent, sequestering the hydrophobic tailregions in the interior of the micelle. Any suitable micelle known inthe art may be used. In some embodiments, micelles may be spherical.Micelles of the present disclosure may be prepared by any suitablemethod known in the art. Examples of suitable methods for preparingmicelles include, without limitation, direct dissolution, and direct ormicroemulsification dialysis, which may encompass preparation bydetergent or water-miscible solvent removal methods.

Solid Lipid Nanoparticles

In some embodiments, a lipid particle of the present disclosure may be asolid lipid nanoparticle. As used herein, a “solid lipid nanoparticle”(SLN) refers to lipid in water emulsions composed of lipids that aregenerally solid at temperatures of at least 50° C., and typicallycontain a solid lipid core matrix that can solubilize lipophilicmolecules. In some variations, solid lipid nanoparticles have a diameterin the range of 10 to 1000 nanometers. Solid lipid nanoparticles mayprotect incorporated active compounds, such as carbohydrates of thepresent disclosure, against chemical degradation and can alsodemonstrate flexibility in modulating the release of such compounds. Thelipid core of solid lipid nanoparticles may be stabilized by surfactants(e.g., emulsifiers). The lipid may typically include triglycerides,diglycerides, monoglycerides, fatty acids, steroids, and/or waxes. Anysuitable solid lipid nanoparticle known in the art may be used. Solidlipid nanoparticles of the present disclosure may be prepared by anysuitable method known in the art. Examples of suitable methods forpreparing solid lipid nanoparticles include, without limitation,microemulsification, high-pressure homogenization, precipitation, andfilm ultrasound dispersion.

Niosomes

In some embodiments, a lipid particle of the present disclosure may be aniosome. As used herein, a “niosome” refers to a vesicular structureformed of a bilayer of non-ionic surfactant molecules and that containsan aqueous core. Niosomes are structurally similar to liposomes inhaving a lamellar structure, however, the materials used to prepareniosomes make them more stable against hydrolytic degradation. Examplesof suitable noisome preparation materials include, without limitation,sterols and one or more non-ionic surfactants. Any suitable niosomeknown in the art may be used. Niosomes of the present disclosure may beprepared by any suitable method known in the art. Examples of suitablemethods for preparing micelles include, without limitation, etherinjection, agitation, bubble method, reverse phase evaporation,sonication, multiple membrane extrusion, and microfluidigation.

Components of the Lipid Particles

Lipid particles of the present disclosure (e.g., liposomes, micelles,solid lipid nanoparticles, and niosomes) contain one or more lipids. Asused herein, the term “lipid” refers to a substance of biological orsynthetic origin that is soluble or partially soluble in organicsolvents or which partitions into a hydrophobic environment when presentin aqueous phase. In some variations, lipids may be divided into atleast three classes: (1) “simple lipids,” which include, withoutlimitation, fats, oils, and waxes; (2) “compound lipids,” which include,without limitation, phospholipids and glycolipids; and (3) “derivedlipids,” which include, without limitation, steroids.

In some variations, the lipid may be a neutral lipid or an amphiphiliclipid. As used herein, the term “neutral lipid” refers to a lipid thatexists either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids may include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.As used herein, the term “amphiphilic lipid” refers to a lipid thatcontains both polar, water-soluble groups and non-polar, water-insolublegroups.

Examples of suitable lipids include, without limitation, bilayer-forminglipids, non-bilayer-forming lipids, amphiphilic lipids,naturally-occurring lipids, phospholipids, glycerolipids, sphingolipids,phosphatidylglycerol, phosphatidic acid, lyso-lipids, fatty acids,sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with ether-linked and ester-linked fatty acids,polymerizable lipids, synthetic lipids, and semi-synthetic lipids.Synthetic or semi-synthetic lipids may be produced via deacylation orreacylation of natural lipids. Suitable features of synthetic andsemi-synthetic lipids include, without limitation, myristoyl, palmitoyl,and stearoyl fatty acids. In some embodiments, lipid particles of thepresent disclosure may contain a mixture of two or more types of lipids.Such mixture may be present at any ratio that is suitable forencapsulating a carbohydrate of the present disclosure and deliveringsuch carbohydrate to a cell interior. In some embodiments, lipidparticles of the present disclosure may contain a lipid selected fromphospholipid, a glycerolipid, a sphingolipid, and any combinationthereof. As disclosed herein, such a lipid has a polar head group and afatty acid tail that may be linked by, for example, an ester linkage oran ether linkage.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain one or more lipids having polar head groups. The lipids maycontain any suitable polar head group known in the art. Examples ofsuitable polar head groups include, without limitation, choline,ethanolamine, serine, glycerol, inositol, and any combination thereof.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) may be,without limitation, lamellar nanostructures that contain an amphiphiliclipid. Examples of suitable amphiphilic lipids include, withoutlimitation, phospholipids, cholesterol, glycolipids, fatty acids, bileacids, saponins, and surfactants.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain one or more phospholipids. Lipid particles of the presentdisclosure may contain any suitable phospholipid known in the art.Examples of suitable phospholipids include, without limitation,phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol(PI), and any combination thereof. In some embodiments, lipid particlesof the present disclosure may contain phosphatidylcholine (PC). In someembodiments, lipid particles of the present disclosure may containphosphatidylethanolamine (PE). In certain embodiments, lipid particlesof the present disclosure may contain phosphatidylcholine (PC) andphosphatidylethanolamine (PE).

In some embodiments, a phospholipid species of the present disclosuremay be present in a lipid particle of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) at aconcentration of up to 80 molar percent. In certain embodiments, aphospholipid of the present disclosure may be present in a lipidparticle of the present disclosure at a concentration of up to 5 molarpercent, up to 10 molar percent, up to 20 molar percent, up to 30 molarpercent, up to 40 molar percent, up to 50 molar percent, up to 60 molarpercent, up to 70 molar percent, or up to 80 molar percent. In certainembodiments, a phospholipid of the present disclosure may be present ina lipid particle of the present disclosure at a concentration of about70 molar percent.

In some embodiments, a lipid particle of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) may bemodified to, for example, include a molecule, such as a stealthmolecule, that shields, or otherwise protects, an encapsulatedcarbohydrate of the present disclosure from degradation, and thatenhances cell permeability, circulation, and/or retention time of thecarbohydrate-containing lipid composition within a cell, tissue, organand/or body of a subject that is administered thecarbohydrate-containing lipid composition. Without wishing to be boundby theory, a lipid particle of the present disclosure dramaticallyenhances cell permeability of carbohydrates, which are otherwiseincapable of traversing the plasma membrane. Accordingly, in someembodiments, a lipid particle of the present disclosure includes amolecule that is capable of minimizing degradation of the lipid particleand/or enhancing retention of the lipid particle when administered to asubject, and/or makes the lipid particle immunotolerant whenadministered to a subject. In some embodiments, the molecule is one ormore of ethylene oxide, an ethylene oxide oligomer, an ethylene oxidepolymer, polyethylene glycol (PEG), or any combination thereof; and/or aPEGylated neutral lipid. As used herein, the term “PEGylated neutrallipid” refers to a neutral lipid, at a selected pH, to which apolyethylene glycol has been conjugated.

As used herein, the terms “ethylene oxide,” “oxirane,” “epoxyethane,”“dimethylene oxide,” and “oxacyclopropane” may be used interchangeablyand refer to a cyclic ether having the formula C₂H₄O.

As used herein, the terms “polyethylene glycol,” “PEG,” “polyethyleneoxide,” “PEO,” “polyoxyethylene,” and “POE” may be used interchangeablyand refer to a polyether compound that is composed of two or moreethylene oxide subunits. “Polyethylene glycol” may be composed ofethylene oxide oligomers (e.g., having from two to nine ethylene oxidemonomer subunits) or ethylene oxide polymers (e.g., having ten or morenine ethylene oxide monomer subunits).

In some embodiments, a lipid particle of the present disclosure may bemodified to include one or more lipids conjugated to polyethylene glycol(PEG) in order to allow the lipid particle to avoid detection by theimmune system of a subject that is administered the lipid particle. Insome embodiments, a lipid particle of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) increasescell permeability for hydrophobic carbohydrates, such as endogenouscarbohydrates of the present disclosure, that are otherwise unable toget through the cell membrane on their own.

Accordingly, in some embodiments, lipid particles of the presentdisclosure (e.g., liposomes, micelles, solid lipid nanoparticles, andniosomes) contain ethylene oxide, oligomers of ethylene oxide and/orpolymers of ethylene oxide. In certain embodiments, lipid particles ofthe present disclosure contain polyethylene glycol (PEG). Alternatively,in some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain, without limitation, polyvinyl pyrrolidone (PVP), polyacrylamide (PAA), G_(M1) ganglioside, cerebroside, and/or sulfates. Withoutwishing to be bound by theory, such modifications can shield lipidparticles from opsonization.

In some embodiments, polyethylene glycol (PEG) may be present in a lipidparticle of the present disclosure (e.g., liposomes, micelles, solidlipid nanoparticles, and niosomes) at a concentration that ranges fromabout 0.5 molar percent to about 20 molar percent. In some embodiments,PEG may be present in a lipid particle of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) at aconcentration of about 0.5 molar percent, about 1 molar percent, about 2molar percent, about 3 molar percent, about 4 molar percent, about 5molar percent, about 6 molar percent, about 7 molar percent, about 8molar percent, about 9 molar percent, about 10 molar percent, about 11molar percent, about 12 molar percent, about 13 molar percent, about 14molar percent, about 15 molar percent, about 16 molar percent, about 17molar percent, about 18 molar percent, about 19 molar percent, or about20 molar percent.

In some embodiments, polyethylene glycol (PEG) may be present in a lipidparticle of the present disclosure (e.g., liposomes, micelles, solidlipid nanoparticles, and niosomes) at a molecular weight concentrationthat ranges from about 200 Da to about 10,000 Da. In some embodiments,polyethylene glycol (PEG) may be present in a lipid particle of thepresent disclosure (e.g., liposomes, micelles, solid lipidnanoparticles, and niosomes) at a molecular weight concentration ofabout 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da,about 700 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500Da, about 2,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da,about 4,000 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about6,000 Da, about 6,500 Da, about 7,000 Da, about 7,500 Da, about 8,000Da, about 8,500 Da, about 9,000 Da, about 9,500 Da, or about 10,000 Da.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain PEG conjugated to a phospholipid of the present disclosure. ThePEG may be conjugated to one or more of phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylglycerol (PG), phosphatidylinositol (PI), and anycombination thereof. In certain embodiments, lipid particles of thepresent disclosure may contain PEG conjugated to phosphatidylcholine(PC). In certain embodiments, lipid particles of the present disclosuremay contain PEG conjugated to phosphatidylethanolamine (PE).

In some embodiments, a phospholipid conjugated to polyethylene glycol(PEG) may be present in a lipid particle of the present disclosure(e.g., liposomes, micelles, solid lipid nanoparticles, and niosomes) ata concentration that ranges from about 0.5 molar percent to about 20molar percent. In some embodiments, a phospholipid conjugated topolyethylene glycol (PEG) may be present in a lipid particle of thepresent disclosure (e.g., liposomes, micelles, solid lipidnanoparticles, and niosomes) at a concentration of about 0.5 molarpercent, about 1 molar percent, about 2 molar percent, about 3 molarpercent, about 4 molar percent, about 5 molar percent, about 6 molarpercent, about 7 molar percent, about 8 molar percent, about 9 molarpercent, about 10 molar percent, about 11 molar percent, about 12 molarpercent, about 13 molar percent, about 14 molar percent, about 15 molarpercent, about 16 molar percent, about 17 molar percent, about 18 molarpercent, about 19 molar percent, or about 20 molar percent. In certainembodiments, a phospholipid conjugated to polyethylene glycol (PEG) maybe present in a lipid particle of the present disclosure at aconcentration of about 2 molar percent.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain one or more sterols. Examples of suitable phospholipids include,without limitation, cholesterol and cholesteryl hemisuccinate (CH),dicetyl phosphate, and Solulan C24.

In some embodiments, a sterol of the present disclosure, such ascholesterol, may be present in a lipid particle of the presentdisclosure (e.g., liposomes, micelles, solid lipid nanoparticles, andniosomes) at a concentration of up to 40 molar percent. In someembodiments, a sterol of the present disclosure, such as cholesterol,may be present in a lipid particle of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) at aconcentration of up to 5 molar percent, up to 10 molar percent, up to 15molar percent, up to 20 molar percent, up to 30 molar percent, or up to40 molar percent. In certain embodiments, a sterol of the presentdisclosure, such as cholesterol, may be present in a lipid particle ofthe present disclosure at a concentration of at most 15 molar percent.In some embodiments, a sterol of the present disclosure, such ascholesterol, may be present in a lipid particle of the presentdisclosure (e.g., liposomes, micelles, solid lipid nanoparticles, andniosomes) at a molar ratio of 1:1 or 2:1.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain one or more fatty acids. In some embodiments, fatty acids of thepresent disclosure may have a carbon chain that ranges in length fromabout 14 carbon atoms to about 18 carbon atoms.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) maycontain one or more neutral lipids. In certain embodiments, a neutrallipid of the present disclosure may be PEGylated conjugated to PEG). Insome embodiments, a PEGylated neutral lipid of the present disclosuremay be present in a lipid particle of the present disclosure at aconcentration that ranges from about 0.2 molar percent to about 20 molarpercent. In some embodiments, a neutral lipid of the present disclosuremay he PEGylated (i.e., conjugated to PEG). In some embodiments, aPEGylated neutral lipid of the present disclosure may be present in alipid particle of the present disclosure at a concentration of about 0.5molar percent, about 1 molar percent, about 2 molar percent, about 3molar percent, about 4 molar percent, about 5 molar percent, about 6molar percent, about 7 molar percent, about 8 molar percent, about 9molar percent, about 10 molar percent, about 11 molar percent, about 12molar percent, about 13 molar percent, about 14 molar percent, about 15molar percent, about 16 molar percent, about 17 molar percent, about 18molar percent, about 19 molar percent, or about 20 molar percent.

In some embodiments, lipid particles of the present disclosure (e.g.,liposomes, micelles, solid lipid nanoparticles, and niosomes) may bemodified to target specific organelles and/or tissues. Any suitablemethod known in the art may be used to target a lipid particle to anorganelle and/or tissue. Suitable examples include, without limitation,modification of the material used for the preparation of the lipidparticle itself (e.g., by utilizing distinct mixtures and molar ratiosof phospholipids such as PC, PS, PI, and PE), covalent attachment of alinker (e.g., peptide chains such as poly-arginine chains andoctadecyl-rhodamine B) to the lipid particle surface, or an antibodymodification. For example, octadecyl-rhodamine B modification can targeta lipid particle to the lysosome of cells. In some embodiments, lipidparticles of the present disclosure (e.g., liposomes, micelles, solidlipid nanoparticles, and niosomes) may be antibody-conjugated liposomes,or immunoliposomes, that may be used to direct encapsulatedcarbohydrates of the present disclosure to diseased tissues and/ororgans.

Carbohydrates

Other aspects of the present disclosure relate to compositionscontaining carbohydrates encapsulated within a lipid particle of thepresent disclosure (e.g., liposomes, micelles, solid lipidnanoparticles, and niosomes). Any suitable carbohydrate known in the artmay be used. Examples of suitable carbohydrates that may be usedinclude, without limitation, monosaccharides, phosphorylatedmonosaccharides, disaccharides, phosphorylated disaccharides,oligosaccharides, phosphorylated oligosaccharides, polysaccharides,phosphorylated polysaccharides, nucleotide sugars, endogenouscarbohydrates, and phosphorylated endogenous carbohydrates.

In some embodiments, the carbohydrate may be present within a lipidparticle of the present disclosure at a concentration that ranges fromabout 0.10 mg per mL of lipid particle to about 10 mg per mL of lipidparticle. In some embodiments, the carbohydrate may be present within alipid particle of the present disclosure at a concentration of about0.10 mg per mL of lipid particle, about 0.20 mg per mL of lipidparticle, about 0.30 mg per mL of lipid particle, about 0.40 mg per mLof lipid particle, about 0.50 mg per mL of lipid particle, about 0.60 mgper mL of lipid particle, about 0.70 mg per mL of lipid particle, about0.80 mg per mL of lipid particle, about 0.90 mg per mL of lipidparticle, about 1 mg per mL of lipid particle, about 1.50 mg per mL oflipid particle, about 2 mg per mL of lipid particle, about 2.50 mg permL of lipid particle, about 3 mg per mL of lipid particle, about 3.50 mgper mL of lipid particle, about 4 mg per mL of lipid particle, about4.50 mg per mL of lipid particle, about 5 mg per mL of lipid particle,5.50 mg per mL of lipid particle, about 6 mg per mL of lipid particle,6.50 mg per mL of lipid particle, about 7 mg per mL of lipid particle,7.50 mg per mL of lipid particle, about 8 mg per mL of lipid particle,8.50 mg per mL of lipid particle, about 9 mg per mL of lipid particle,about 9.50 mg per mL of lipid particle, or about 10 mg per mL of lipidparticle. In certain embodiments, a carbohydrate of the presentdisclosure may be present within a lipid particle of the presentdisclosure at a concentration of about 1 mg per mL of lipid particle.

In some embodiments, a carbohydrate of the present disclosure is anendogenous carbohydrate. As used herein, “endogenous carbohydrate”refers to a carbohydrate that is found as a natural product in a subject(including, for example, a human). It should be understood, however,that an endogenous carbohydrate may be either (i) naturally produced bya subject (including, for example, a human) and extracted from theliving cells of such subject, or (ii) synthetically made. In someembodiments, the endogenous carbohydrate is produced in vivo by asubject (including, for example, human). In other embodiments, theendogenous carbohydrate is naturally produced by a cell derived from asubject (including, for example, human), such as a cultured cell line.Thus, the source of such endogenous carbohydrates may include, withoutlimitation, a synthetic source (e.g., chemical synthesis) or a naturalsource (e.g., extraction, isolation, or purification from a subject orcell that naturally produces the endogenous carbohydrate or arecombinant cell, such as a bacterial cell, that has been geneticallyengineered to produce the endogenous carbohydrate). Examples ofendogenous carbohydrates may include, without limitation, carbohydratesinvolved in protein and lipid glycosylation.

Examples of suitable endogenous carbohydrates include, withoutlimitation, a monosaccharide, a phosphorylated monosaccharide, adisaccharide, a phosphorylated disaccharide, an oligosaccharide, aphosphorylated oligosaccharide, a polysaccharide, a phosphorylatedpolysaccharide, mannose, a phosphorylated mannose, a mannofuranose, aphosphorylated mannofuranose, a mannopyranos, a phosphorylatedmannopyranos, mannose-1-phosphate, a nucleotide sugar, a uridinediphosphate, a guanine diphosphate, a cytosine monophosphate, fucose,GDP-fucose, a sialic acid, CMP-sialic acid, N-acetylneuraminic acid(Neu5Ac), and CMP-Neu5Ac.

In other embodiments, the carbohydrate is a dolichol pyrophosphatelinked oligosaccharide, Glc3Man9GlcNAc2-PP-Dol, where Glc=glucose,Man=mannose, GlcNAc=N-Acetylglucosamine, P=phosphate, and Dol=dolicholwith a chain length that includes, but not limited to, 14-18 isopreneunits. Any suitable method known in the art for preparing dolicholpyrophosphate linked oligosaccharides may be used. In one example, achemoenzymatic approach may be used to synthesize dolichol pyrophosphatelinked oligosaccharide (e.g., Wang Z. et al., Science, 2013, 341,379-383 and Weerapana E. et al., J. Am. Chem. Soc., 2005, 127,13766-13767).

Other aspects of the present disclosure relate to compositionscontaining glycolipids encapsulated within a lipid particle of thepresent disclosure. Any suitable glycolipid known in the art may beused. Glycolipids of the present disclosure include, without limitation,any class and molecular weight of lipids and carbohydrates.

In some embodiments, carbohydrates and glycolipids of the presentdisclosure may be integrated into lipid particles of the presentdisclosure, such as liposomes, that may be designed for specifictargeting to a cell interior, the ER of a cell, or the Golgi of a cell.

Pharmaceutical Compositions

Compositions of the present disclosure containing a lipid particle ofthe present disclosure and a carbohydrate of the present disclosureencapsulated in the lipid particle can be incorporated into a variety offormulations for therapeutic use (e.g., by administration) or in themanufacture of a medicament (e.g., for delivering a carbohydrate of thepresent disclosure to a subject in need thereof and/or cell interior ofa subject in need thereof and/or for treating or preventing a disease ordisorder such as a congenital disorder of glycosylation (CDG) in asubject in need thereof) by combining the composition with appropriatecarriers (including, for example, pharmaceutically acceptable carriersor diluents), and may be formulated, without limitation, intopreparations in liquid, aerosolized, semisolid, or powder forms.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or subjectbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers include,without limitation, buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Examples of suitable formulations include, without limitation,solutions, injections, inhalants, microspheres, aerosols, gels,ointments, creams, lotions, powders, dry vescicular powders, tablets,and capsules. Pharmaceutical compositions can include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers ofdiluents, which are vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents include, without limitation, distilled water, bufferedwater, physiological saline, PBS, Ringer's solution, dextrose solution,and Hank's solution. A pharmaceutical composition or formulation of thepresent disclosure can further include, without limitation, othercarriers or non-toxic, nontherapeutic, nonimmunogenic stabilizers, andexcipients. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents. Apharmaceutical composition of the present disclosure can also includeany of a variety of stabilizing agents, such as an antioxidant forexample.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Pharmaceutical Dosages

Pharmaceutical compositions of the present disclosure containing acomposition containing a lipid particle of the present disclosure and acarbohydrate of the present disclosure encapsulated by the lipidparticle may be used (e.g., administered to a subject in need oftreatment with a carbohydrate of the present disclosure, such as a humanindividual) in accord with known methods, such as oral administration,intravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,intracranial, intraspinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

Dosages and desired concentration of pharmaceutical compositions of thepresent disclosure may vary depending on the particular use envisioned.The determination of the appropriate dosage or route of administrationis well within the skill of an ordinary artisan. Animal experimentsprovide reliable guidance for the determination of effective doses forhuman therapy. Interspecies scaling of effective doses can be performedfollowing the principles described in Mordenti, J. and Chappell, W. “TheUse of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics andNew Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989,pp. 42-46.

For in vivo administration of any of the compositions of the presentdisclosure containing a lipid particle of the present disclosure and acarbohydrate of the present disclosure encapsulated by the lipidparticle, normal dosage amounts may vary from 10 ng/kg up to 100 mg/kgof a subject's body weight per day.

Administration of a composition of the present disclosure containing alipid particle of the present disclosure and a carbohydrate of thepresent disclosure encapsulated by the lipid particle can be continuousor intermittent, depending, for example, on the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners.

It is within the scope of the present disclosure that differentformulations will be effective for different treatments and differentdisorders, and that administration intended to treat a specific organ ortissue may necessitate delivery in a manner different from that toanother organ or tissue. Moreover, dosages may be administered by one ormore separate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Thus, in some variations, the compositions provided herein may bechronically or intermittently administered to a subject (including, forexample, a human) in need thereof. “Chronic” administration refers toadministration of the medicament(s) in a continuous as opposed to acutemode, so as to maintain the initial therapeutic effect (activity) for anextended period of time. “Intermittent” administration refers totreatment that is not consecutively done without interruption, butrather is cyclic in nature.

Therapeutic Uses

The present disclosure provides compositions containing a lipid particleof the present disclosure and a carbohydrate of the present disclosureencapsulated by the lipid particle that are capable of delivering thecarbohydrate into the interior of a cell. These compositions are usefulfor delivering carbohydrates of the present disclosure to a subject inneed of such carbohydrates.

As used herein, the term “subject” refers to a mammal, such as a human,domestic animal, such as a feline or canine subject, farm animal (e.g.,bovine, equine, caprine, ovine, and porcine subject), wild animal(whether in the wild or in a zoological garden), research animal, suchas mouse, rat, rabbit, goat, sheep, pig, dog, and cat, and birds. In oneembodiment, the subject is a human.

In some variations, the subject may be at risk. For example, in onevariation, the subject is an at risk human. As used herein, a subject“at risk” of developing a particular disease, disorder, or condition,such as a congenital disorder of glycosylation, may or may not havedetectable disease or symptoms of disease, and may or may not havedisplayed detectable disease or symptoms of disease prior to thetreatment methods described herein. “At risk” denotes that an individualhas risk factors, which are measurable parameters that correlate withdevelopment of a particular disease, disorder, or condition, as known inthe art. A subject having one or more of these risk factors has a higherprobability of developing a particular disease, disorder, or conditionsuch as a congenital disorder of glycosylation, than a subject withoutone or more of these risk factors.

In some embodiments, compositions containing a lipid particle of thepresent disclosure and a carbohydrate of the present disclosureencapsulated by the lipid particle may also be used for deliveringcarbohydrates of the present disclosure to the cell interior of asubject in need thereof. In certain embodiments, compositions containinga lipid particle of the present disclosure and a carbohydrate of thepresent disclosure encapsulated by the lipid particle may be used forpreventing, reducing risk, or treating a disease or disorder, such as acongenital disorder of glycosylation (CDG) in a subject in need thereof.

Accordingly, as disclosed herein, compositions of the present disclosurecontaining a lipid particle of the present disclosure and a carbohydrateof the present disclosure encapsulated by the lipid particle may be usedfor delivering a carbohydrate of the present disclosure to a subject inneed thereof, for delivering a carbohydrate of the present disclosure tothe cell interior of a subject in need thereof, and for treating,preventing, or reducing risk of a disease or disorder, such as acongenital disorder of glycosylation (CDG) in a subject in need thereof.In some embodiments, the subject has such a disease or disorder. In someembodiments, the subject is a human having such a disease or disorder.

As used herein, the term “congenital disorders of glycosylation” (CDG)refers to a group of genetic disorders that result in errors ofmetabolism in which glycosylation of a variety of tissue proteins and/orlipids is deficient or defective. Congenital disorders of glycosylationmay also be known as CDG syndromes. CDG syndromes may often causeserious, occasionally fatal, malfunction of several different organsystems, such as the nervous system, brain, muscles, and intestines, inaffected infants. Manifestations of CDG syndromes may range from severedevelopmental delay and hypotonia beginning in infancy, to hypoglycemiaand protein-losing enteropathy with normal development. Developmentaldelay can be a common initial indication for a CDG diagnosis. One of themost common subtype of CDG syndromes is CDG-Ia (also known as PMM2-CDG)where the genetic defect leads to the loss of phosphomannomutase 2,which is the enzyme responsible for the conversion ofmannose-6-phosphate into mannose-1-phosphate.

CDG syndromes may be classified as type I (CDG-I) and type II (CDG-II).Such classification may depend on the nature and location of thebiochemical defect in the metabolic pathway relative to the action ofoligosaccharyltransferase. Methods for screening for CDG subtype mayinclude the analysis of transferrin glycosylation status by, forexample, isoelectric focusing or ESI-MS. Examples of CDG type I include,without limitation, Ia (PMM2-CDG), Ib (MPI-CDG), Ic (ALG6-CDG) , Id(ALG3-CDG), Ie (DPM1-CDG), If (MPDU1-CDG), Ig (ALG12-CDG), Ih(ALG8-CDG), Ii (ALG2-CDG), Ij (DPAGT1-CDG), Ik (ALG1-CDG), 1L(ALG9-CDG), Im (DOLK-CDG), In (RFT1-CDG), Io (DPM3-CDG), Ip (ALG11-CDG),Iq (SRD5A3-CDG), Ir (DDOST-CDG), DPM2-CDG, TUSC3-CDG, MAGT1-CDG,DHDDS-CDG, and I/IIx. Examples of CDG type II include, withoutlimitation, IIa (MGAT2-CDG), IIb (GCS1-CDG), IIc (SLC335C1-CDG), IId(B4GALT1-CDG), IIe (COG7-CDG), IIf (SLC35A1-CDG), IIg (COG1-CDG), IIh(COG8-CDG), IIi (COG5-CDG), IIj (COG4-CDG), IIL (COG6-CDG),ATP6V0A2-CDG, MAN1B1-CDG, and ST3GAL3-CDG.

Congenital disorders of glycosylation (CDG) that may be treated withcompositions of the present disclosure containing a lipid particle ofthe present disclosure and a carbohydrate of the present disclosureencapsulated by the lipid particle include, without limitation, Ia(PMM2-CDG), Ib (MPI-CDG), Ic (ALG6-CDG) , Id (ALG3-CDG), Ie (DPM1-CDG),If (MPDU1-CDG), Ig (ALG12-CDG), Ih (ALG8-CDG), Ii (ALG2-CDG), Ij(DPAGT1-CDG), Ik (ALG1-CDG), 1L (ALG9-CDG), Im (DOLK-CDG), In(RFT1-CDG), Io (DPM3-CDG), Ip (ALG11-CDG), Iq (SRD5A3-CDG), Ir(DDOST-CDG), DPM2-CDG, TUSC3-CDG, MAGT1-CDG, DHDDS-CDG, I/IIx, IIa(MGAT2-CDG), IIb (GCS1-CDG), IIc (SLC335C1-CDG), IId (B4GALT1-CDG), IIe(COG7-CDG), IIf (SLC35A1-CDG), IIg (COG1-CDG), IIh (COG8-CDG), IIi(COG5-CDG), IIj (COG4-CDG), IIL (COG6-CDG), ATP6V0A2-CDG, MAN1B1-CDG,and ST3GAL3-CDG.

Accordingly, the compositions of the present disclosure containing alipid particle of the present disclosure and a carbohydrate of thepresent disclosure encapsulated by the lipid particle may be used totreat, prevent, or improve one or more symptoms of a congenital disorderof glycosylation (CDG) of the present disclosure. In some embodiments,the present disclosure provides methods of treating, preventing, orimproving one or more symptoms in subjects having a congenital disorderof glycosylation (CDG) disorder of the present disclosure byadministering to the subject a composition of the present disclosurecontaining a lipid particle of the present disclosure and a carbohydrateof the present disclosure encapsulated by the lipid particle to, forexample, deliver a carbohydrate to the subject to correct, or otherwiseimprove protein and/or lipid glycosylation in the subject.

In some embodiments, the present disclosure provides methods fortreating, preventing, or improving one or more symptoms of a congenitaldisorder of glycosylation (CDG) to a subject in need thereof, byadministering to the subject a therapeutically effective amount of acomposition comprising a lipid particle; and an endogenous carbohydrateencapsulated in the lipid particle. In some embodiments, the presentdisclosure provides methods for treating, preventing, or improving oneor more symptoms of a congenital disorder of glycosylation (CDG) to asubject in need thereof, by administering to the subject atherapeutically effective amount of a composition comprising a lipidparticle comprising ethylene oxide; and a carbohydrate encapsulated inthe lipid particle. In some embodiments, the present disclosure providesmethods for treating, preventing, or improving one or more symptoms of acongenital disorder of glycosylation (CDG) to a subject in need thereof,by administering to the subject a therapeutically effective amount of acomposition comprising a lipid particle; and a carbohydrate encapsulatedin the lipid particle, wherein the lipid particle comprises choline,ethanolamine, glycerol, inositol, or any combination thereof. In someembodiments, the present disclosure provides methods for treating,preventing, or improving one or more symptoms of a congenital disorderof glycosylation (CDG) to a subject in need thereof, by administering tothe subject a therapeutically effective amount of a compositioncomprising a liposome; and mannose-1-phosphate encapsulated in theliposome, wherein the liposome comprises cholesterol andphosphatidylethanolamine (PE) attached to polyethylene glycol (PEG). Insome embodiments, the congenital disorder of glycosylation (CDG) may bea CDG type I disorder, a CDG-Ia disorder, a CDG type II disorder, aCDG-IIc disorder, or a CDG-IIf disorder. In certain embodiments, thecongenital disorder of glycosylation (CDG) is a CDG-Ia disorder. In someembodiments, the composition is administered orally, dermally, nasally,or intravenously.

As used herein, the term “treatment” or “treating” is an approach forobtaining beneficial or desired results including clinical results.Beneficial or desired clinical results may include one or more of thefollowing: a) inhibiting the disease or condition (e.g., decreasing oneor more symptoms resulting from the disease or condition, and/ordiminishing the extent of the disease or condition); b) slowing orarresting the development of one or more clinical symptoms associatedwith the disease or condition (e.g., stabilizing the disease orcondition, preventing or delaying the worsening or progression of thedisease or condition, and/or preventing or delaying the spread of thedisease or condition); and/or c) relieving the disease, that is, causingthe regression of clinical symptoms (e.g., ameliorating the diseasestate, providing partial or total remission of the disease or condition,enhancing effect of another medication, delaying the progression of thedisease, increasing the quality of life, and/or prolonging survival.

As used herein, the term “prevention” or “preventing” refers to anytreatment of a disease or condition that causes the clinical symptoms ofthe disease or condition not to develop. Compounds may, in someembodiments, be administered to a subject (including a human) who is atrisk or has a family history of the disease or condition.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disease, disorder, or condition, such as a congenitaldisorder of glycosylation. A therapeutically effective amount herein mayvary according to factors such as the disease state, age, sex, andweight of the subject, and the ability of the lipid compositions of thepresent disclosure to elicit a desired response in the subject. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the lipid compositions of the present disclosureare outweighed by the therapeutically beneficial effects.

In other aspects of the present disclosure, compositions of the presentdisclosure containing a lipid particle of the present disclosure and acarbohydrate of the present disclosure encapsulated by the lipidparticle may be used for delivering a carbohydrate to a subject in needthereof. Accordingly, in some embodiments, the present disclosureprovides methods for delivering a carbohydrate to a subject in needthereof, by administering to the subject a therapeutically effectiveamount of a composition comprising a lipid particle; and an endogenouscarbohydrate encapsulated in the lipid particle. In some embodiments,the present disclosure provides methods for delivering a carbohydrate toa subject in need thereof, by administering to the subject atherapeutically effective amount of a composition comprising a lipidparticle comprising ethylene oxide; and a carbohydrate encapsulated inthe lipid particle. In some embodiments, the present disclosure providesmethods for delivering a carbohydrate to a subject in need thereof, byadministering to the subject a therapeutically effective amount of acomposition comprising a lipid particle; and a carbohydrate encapsulatedin the lipid particle, wherein the lipid particle comprises choline,ethanolamine, glycerol, inositol, or any combination thereof. In someembodiments, the present disclosure provides methods for delivering acarbohydrate to a subject in need thereof, by administering to thesubject a therapeutically effective amount of a composition comprising aliposome; and mannose-1-phosphate encapsulated in the liposome, whereinthe liposome comprises cholesterol and phosphatidylethanolamine (PE)attached to polyethylene glycol (PEG). In some embodiments, the subjecthas a congenital disorder of glycosylation (CDG). In some embodiments,the congenital disorder of glycosylation (CDG) may be a CDG type Idisorder, a CDG-Ia disorder, a CDG type II disorder, a CDG-IIc disorder,and a CDG-IIf disorder. In certain embodiments, the congenital disorderof glycosylation (CDG) is a CDG-Ia disorder.

In other aspects of the present disclosure, compositions of the presentdisclosure containing a lipid particle of the present disclosure and acarbohydrate of the present disclosure encapsulated by the lipidparticle may be used for delivering a carbohydrate to a cell interior ofa subject in need thereof. Accordingly, in some embodiments, the presentdisclosure provides methods for delivering a carbohydrate to a cellinterior of a subject in need thereof, by administering to the subject atherapeutically effective amount of a composition comprising a lipidparticle; and an endogenous carbohydrate encapsulated in the lipidparticle, wherein the administered composition traverses the cell plasmamembrane thereby delivering the carbohydrate to the cell interior. Insome embodiments, the present disclosure provides methods for deliveringa carbohydrate to a cell interior of a subject in need thereof, byadministering to the subject a therapeutically effective amount of acomposition comprising a lipid particle comprising ethylene oxide; and acarbohydrate encapsulated in the lipid particle, wherein theadministered composition traverses the cell plasma membrane therebydelivering the carbohydrate to the cell interior. In some embodiments,the present disclosure provides methods for delivering a carbohydrate toa cell interior of a subject in need thereof, by administering to thesubject a therapeutically effective amount of a composition comprising alipid particle; and a carbohydrate encapsulated in the lipid particle,wherein the lipid particle comprises choline, ethanolamine, glycerol,inositol, or any combination thereof, and wherein the administeredcomposition traverses the cell plasma membrane thereby delivering thecarbohydrate to the cell interior. In some embodiments, the presentdisclosure provides methods for delivering a carbohydrate to a cellinterior of a subject in need thereof, by administering to the subject atherapeutically effective amount of a composition comprising a liposome;and mannose-1-phosphate encapsulated in the liposome, wherein theliposome comprises cholesterol and phosphatidylethanolamine (PE)attached to polyethylene glycol (PEG), and wherein the administeredcomposition traverses the cell plasma membrane thereby delivering thecarbohydrate to the cell interior. In some embodiments, the subject hasa congenital disorder of glycosylation (CDG). In some embodiments, thecongenital disorder of glycosylation (CDG) may be a CDG type I disorder,a CDG-Ia disorder, a CDG type II disorder, a CDG-IIc disorder, and aCDG-IIf disorder. In certain embodiments, the congenital disorder ofglycosylation (CDG) is a CDG-Ia disorder. In some embodiments,administration of the composition induces at least a 0.05-fold to atleast a 2-fold increase in cellular production of higher-orderlipid-linked oligosaccharides in the subject, as compared to cellularproduction of higher-order lipid-linked oligosaccharides in the subjectin the absence of the composition. In certain embodiments, thehigher-order lipid-linked oligosaccharides comprise Man4GlcNAc2,Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or anycombination thereof. In some embodiments, the composition isadministered orally, topically, dermally, nasally, intravenously,intramuscularly, intraperitoneally, intracerobrospinally,intracranially, intraspinally, subcutaneously, intra-articularly,intrasynovialy, or intrathecaly.

Articles of Manufacture and Kits

The present disclosure also provides articles of manufacture and/or kitscontaining a composition of the present disclosure containing a lipidparticle of the present disclosure and a carbohydrate of the presentdisclosure encapsulated by the lipid particle. Articles of manufactureand/or kits of the present disclosure may include one or more containerscomprising a purified composition of the present disclosure. Suitablecontainers may include, without limitation, bottles, vials, syringes,and IV solution bags. The containers may be formed from a variety ofmaterials such as glass or plastic. In some embodiments, the articles ofmanufacture and/or kits further include instructions for use inaccordance with any of the methods of the present disclosure. In someembodiments, these instructions comprise a description of administrationof the composition containing a lipid particle of the present disclosureand a carbohydrate of the present disclosure encapsulated by the lipidparticle to deliver the carbohydrate to a subject in need thereof, todeliver the carbohydrate to a cell interior of a subject in needthereof, or to treat a congenital disorder of glycosylation (CDG) to asubject in need thereof, according to any of the methods of the presentdisclosure. In some embodiments, the instructions comprise a descriptionof how to detect a congenital disorder of glycosylation (CDG), forexample in a subject, in a tissue sample, or in a cell. The article ofmanufacture and/or kit may further comprise a description of selecting asubject suitable for treatment based on identifying whether that subjecthas the disease and the stage of the disease.

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the articles of manufactureand/or kits of the present disclosure are typically written instructionson a label or package insert (e.g., a paper sheet included in thearticle of manufacture and/or kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

The label or package insert indicates that the composition is used fordelivering a carbohydrate and/or treating, e.g., a congenital disorderof glycosylation (CDG). Instructions may be provided for practicing anyof the methods described herein.

The articles of manufacture and/or kits of the present disclosure may bein suitable packaging. Suitable packaging includes, without limitation,vials, bottles, jars, and flexible packaging (e.g., sealed Mylar orplastic bags). Also contemplated are packages for use in combinationwith a specific device, such as an inhaler, nasal administration device(e.g., an atomizer) or an infusion device such as a minipump. An articleof manufacture and/or kit may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The container mayalso have a sterile access port (e.g., the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is a carbohydrate capable of treating a congenital disorderof glycosylation (CDG) and/or improving one or more symptoms thereof.The container may further comprise a second pharmaceutically activeagent.

Articles of manufacture and/or kits may optionally provide additionalcomponents such as buffers and interpretive information. Normally, thearticle of manufacture and/or kit comprises a container and a label orpackage insert(s) on or associated with the container.

Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. For the purpose of the present disclosure, the followingterms are defined.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, “about x” includes and describes “x” per se. In someembodiments, the term “about” when used in association with ameasurement, or used to modify a value, a unit, a constant, or a rangeof values, refers to variations of +/−2%.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to a “lipid particle” is a referenceto from one to many lipid particles, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

Enumerated Embodiments

The following enumerated embodiments are representative of some aspectsof the invention.

-   1. A composition comprising:

a lipid particle; and

an endogenous carbohydrate encapsulated in the lipid particle.

-   2. The composition of embodiment 1, wherein the lipid particle    comprises a molecule that is capable of minimizing degradation of    the lipid particle and/or enhancing retention of the lipid particle    when administered to a subject, and/or makes the lipid particle    immunotolerant when administered to a subject.-   3. The composition of embodiment 2, wherein the molecule is ethylene    oxide, an ethylene oxide oligomer, an ethylene oxide polymer,    polyethylene glycol (PEG), or any combination thereof.-   4. The composition of embodiment 2, wherein the molecule is ethylene    oxide.-   5. The composition of embodiment 2, wherein the molecule is an    ethylene oxide oligomer or an ethylene oxide polymer.-   6. The composition of embodiment 2, wherein the molecule is    polyethylene glycol (PEG).-   7. The composition of any one of embodiments 1-6, wherein the lipid    particle further comprises a lipid selected from the group    consisting of a phospholipid, a glycerolipid, a sphingolipid, and    any combination thereof; optionally wherein the lipid comprises    choline, ethanolamine, serine, glycerol, inositol, or any    combination thereof.-   8. The composition of any one of embodiments 1-7, wherein the lipid    particle further comprises a lipid having a polar head group.-   9. The composition of embodiment 8, wherein the polar head group is    selected from the group consisting of choline, ethanolamine, serine,    glycerol, inositol, and any combination thereof.-   10. The composition of any one of embodiments 1-7, wherein the lipid    particle further comprises a phospholipid.-   11. The composition of embodiment 10, wherein the phospholipid    comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE),    phosphatidylserine (PS), phosphatidylglycerol (PG),    phosphatidylinositol (PI), or any combination thereof.-   12. The composition of any one of embodiments 1-11, wherein the    lipid particle further comprises a sterol.-   13. The composition of embodiment 12, wherein the sterol is    cholesterol.-   14. The composition of embodiment 13, wherein the cholesterol is    present at a concentration of at most 15 molar percent.-   15. The composition of any one of embodiments 1-14, wherein the    lipid particle further comprises a fatty acid.-   16. The composition of any one of embodiments 1-15, wherein the    lipid particle further comprises a PEGylated neutral lipid.-   17. The composition of any one of embodiments 1-16, wherein the    lipid particle is a lamellar nanostructure comprising an amphiphilic    lipid.-   18. The composition of any one of embodiments 1-17, wherein the    lipid particle is selected from the group consisting of a liposome,    a micelle, a solid lipid nanoparticle, and a niosome.-   19. The composition of any one of embodiments 1-17, wherein the    lipid particle is a liposome.-   20. The composition of embodiment 17, wherein the liposome comprises    polyethylene-glycol (PEG).-   21. The composition of embodiment 20, wherein the liposome is a    stealth liposome.-   22. The composition of embodiment 21, wherein the stealth liposome    is immunotolerant.-   23. The composition of any one of embodiments 19-22, wherein the    liposome further comprises a phospholipid selected from the group    consisting of phosphatidylcholine (PC), phosphatidylethanolamine    (PE), phosphatidylserine (PS), phosphatidylglycerol (PG),    phosphatidylinositol (PI), and any combination thereof.-   24. The composition of any one of embodiments 19-22, wherein the    liposome further comprises phosphatidylcholine (PC) and    phosphatidylethanolamine (PE).-   25. The composition of any one of embodiments 19-24, wherein the    liposome further comprises phosphatidylcholine (PC) at a    concentration of at least 70 molar percent.-   26. The composition of any one of embodiments 19-25, wherein the    liposome further comprises a PEGylated neutral lipid.-   27. The composition of embodiment 26, wherein the PEGylated neutral    lipid is present in the lipid particle at a concentration of at    least 1 molar percent.-   28. The composition of any one of embodiments 19-27, wherein the    liposome further comprises cholesterol.-   29. The composition of any one of embodiments 19-28, wherein the    liposome further comprises a fatty acid.-   30. The composition of embodiment 29, wherein the fatty acid    comprises a carbon chain that ranges in length from 14-18 carbon    atoms.-   31. The composition of any one of embodiments 1-17, wherein the    lipid particle is a micelle.-   32. The composition of any one of embodiments 1-17, wherein the    lipid particle is a solid lipid nanoparticle.-   33. The composition of any one of embodiments 1-17, wherein the    lipid particle is a niosome.-   34. The composition of any one of embodiments 1-33, wherein the    endogenous carbohydrate is selected from the group consisting of a    monosaccharide, a phosphorylated monosaccharide, a disaccharide, a    phosphorylated disaccharide, an oligosaccharide, a phosphorylated    oligosaccharide, a polysaccharide, a phosphorylated polysaccharide,    mannose, a phosphorylated mannose, a mannofuranose, a phosphorylated    mannofuranose, a mannopyranos, a phosphorylated mannopyranos,    mannose-1-phosphate, a nucleotide sugar, a uridine diphosphate, a    guanine diphosphate, a cytosine monophosphate, fucose, GDP-fucose, a    sialic acid, CMP-sialic acid, N-acetylneuraminic acid (Neu5Ac), and    CMP-Neu5Ac.-   35. The composition of any one of embodiments 1-33, wherein the    endogenous carbohydrate is mannose-1-phosphate.-   36. The composition of any one of embodiments 1-33, wherein the    endogenous carbohydrate is GDP-fucose.-   37. The composition of any one of embodiments 1-33, wherein the    endogenous carbohydrate is CMP-sialic acid.-   38. The composition of any one of embodiments 1-37, wherein the    lipid particle has an average particle size that ranges from 0.05 to    0.5 microns in diameter.-   39. The composition of any one of embodiments 1-38, wherein the    lipid particle is capable of delivering the endogenous carbohydrate    to a cell interior.-   40. The composition of any one of embodiments 1-39, wherein the    composition when administered to a subject in need thereof induces    at least a 0.05-fold to at least a 3-fold increase in cellular    production of higher-order lipid-linked oligosaccharides in the    subject, as compared to cellular production of higher-order    lipid-linked oligosaccharides in the subject in the absence of    administering the composition to the subject.-   41. The composition of embodiment 40, wherein the higher-order    lipid-linked oligosaccharides comprise Man4GlcNAc2, Man5GlcNAc2,    Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or any    combination thereof.-   42. A composition comprising:

a lipid particle comprising ethylene oxide, an ethylene oxide oligomer,an ethylene oxide polymer, polyethylene glycol (PEG), or any combinationthereof; and

a carbohydrate encapsulated in the lipid particle.

-   43. The composition of embodiment 42, wherein the carbohydrate is an    endogenous carbohydrate.-   44. The composition of embodiment 43, wherein the endogenous    carbohydrate is selected from the group consisting of a    monosaccharide, a phosphorylated monosaccharide, a disaccharide, a    phosphorylated disaccharide, an oligosaccharide, a phosphorylated    oligosaccharide, a polysaccharide, a phosphorylated polysaccharide,    mannose, a phosphorylated mannose, a mannofuranose, a phosphorylated    mannofuranose, a mannopyranos, a phosphorylated mannopyranos,    mannose-1-phosphate, a nucleotide sugar, a uridine diphosphate, a    guanine diphosphate, a cytosine monophosphate, fucose, GDP-fucose, a    sialic acid, CMP-sialic acid, N-acetylneuraminic acid (Neu5Ac), and    CMP-Neu5Ac.-   45. The composition of embodiment 42, wherein the carbohydrate is    mannose-1-phosphate.-   46. The composition of embodiment 42, wherein the carbohydrate is    GDP-fucose.-   47. The composition of embodiment 42, wherein the carbohydrate is    CMP-sialic acid.-   48. The composition of any one of embodiments 42-47, wherein the    lipid particle comprises an ethylene oxide oligomer.-   49. The composition of any one of embodiments 42-47, wherein the    lipid particle comprises an ethylene oxide polymer.-   50. The composition of any one of embodiments 42-47, wherein the    lipid particle comprises polyethylene glycol (PEG).-   51. The composition of any one of embodiments 42-50, wherein the    lipid particle further comprises choline, ethanolamine, serine,    glycerol, inositol, or any combination thereof.-   52. The composition of any one of embodiments 42-51, wherein the    lipid particle further comprises a lipid selected from the group    consisting of a phospholipid, a glycerolipid, a sphingolipid, and    any combination thereof.-   53. The composition of embodiment 52, wherein the lipid comprises a    polar head group.-   54. The composition of embodiment 53, wherein the polar head group    is selected from the group consisting of choline, ethanolamine,    serine, glycerol, inositol, and any combination thereof.-   55. The composition of any one of embodiments 42-52, wherein the    lipid particle further comprises a phospholipid.-   56. The composition of embodiment 55, wherein the phospholipid is    selected from the group consisting of phosphatidylcholine (PC),    phosphatidylethanolamine (PE), phosphatidylserine (PS),    phosphatidylglycerol (PG), phosphatidylinositol (PI), and any    combination thereof.-   57. The composition of any one of embodiments 42-56, wherein the    lipid particle further comprises a sterol.-   58. The composition of embodiment 57, wherein the sterol is    cholesterol.-   59. The composition of embodiment 58, wherein the cholesterol is    present at a concentration of at most 15 molar percent.-   60. The composition of any one of embodiments 42-59, wherein the    lipid particle further comprises a fatty acid.-   61. The composition of any one of embodiments 42-60, wherein the    lipid particle further comprises a PEGylated neutral lipid.-   62. The composition of any one of embodiments 42-61, wherein the    lipid particle is a lamellar nanostructure comprising an amphiphilic    lipid.-   63. The composition of any one of embodiments 42-62, wherein the    lipid particle is selected from the group consisting of a liposome,    a micelle, a solid lipid nanoparticle, and a niosome.-   64. The composition of any one of embodiments 42-62, wherein the    lipid particle is a liposome.-   65. The composition of embodiment 64, wherein the liposome comprises    polyethylene-glycol (PEG).-   66. The composition of embodiment 65, wherein the liposome is a    stealth liposome.-   67. The composition of embodiment 66, wherein the stealth liposome    is immunotolerant.-   68. The composition of any one of embodiments 64-67, wherein the    liposome further comprises a phospholipid selected from the group    consisting of phosphatidylcholine (PC), phosphatidylethanolamine    (PE), phosphatidylserine (PS), phosphatidylglycerol (PG),    phosphatidylinositol (PI), and any combination thereof.-   69. The composition of any one of embodiments 64-67, wherein the    liposome further comprises phosphatidylcholine (PC) and    phosphatidylethanolamine (PE).-   70. The composition of any one of embodiments 64-69, wherein the    liposome comprises phosphatidylcholine (PC) at a concentration of at    least 70 molar percent.-   71. The composition of any one of embodiments 64-70, wherein the    liposome further comprises a PEGylated neutral lipid.-   72. The composition of embodiment 71, wherein the PEGylated neutral    lipid is present in the lipid particle at a concentration of at    least 1 molar percent.-   73. The composition of any one of embodiments 64-72, wherein the    liposome further comprises cholesterol.-   74. The composition of any one of embodiments 64-73, wherein the    liposome further comprises a fatty acid.-   75. The composition of embodiment 74, wherein the fatty acid    comprises a carbon chain that ranges in length from 14-18 carbon    atoms.-   76. The composition of any one of embodiments 42-62, wherein the    lipid particle is a micelle.-   77. The composition of any one of embodiments 42-62, wherein the    lipid particle is a solid lipid nanoparticle.-   78. The composition of any one of embodiments 42-62, wherein the    lipid particle is a niosome.-   79. The composition of any one of embodiments 42-78, wherein the    lipid particle has an average particle size that ranges from 0.05 to    0.5 microns in diameter.-   80. The composition of any one of embodiments 42-79, wherein the    lipid particle is capable of delivering the carbohydrate to a cell    interior.-   81. The composition of any one of embodiments 42-80, wherein the    composition when administered to a subject induces at least a    0.05-fold to at least a 3-fold increase in cellular production of    higher-order lipid-linked oligosaccharides in the subject, as    compared to cellular production of higher-order lipid-linked    oligosaccharides in the subject in the absence of the composition.-   82. The composition of embodiment 81, wherein the higher-order    lipid-linked oligosaccharides comprise Man4GlcNAc2, Man5GlcNAc2,    Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or any    combination thereof.-   83. A composition comprising:

a lipid particle; and

a carbohydrate encapsulated in the lipid particle,

wherein the lipid particle comprises choline, ethanolamine, glycerol,inositol, or any combination thereof.

-   84. The composition of embodiment 83, wherein the lipid particle    further comprises a molecule that is capable of minimizing    degradation of the lipid particle and/or enhancing retention of the    lipid particle when administered to a subject, and/or makes the    lipid particle immunotolerant when administered to a subject.-   85. The composition of embodiment 83 or 84, wherein the carbohydrate    is an endogenous carbohydrate.-   86. The composition of embodiment 85, wherein the endogenous    carbohydrate is selected from the group consisting of a    monosaccharide, a phosphorylated monosaccharide, a disaccharide, a    phosphorylated disaccharide, an oligosaccharide, a phosphorylated    oligosaccharide, a polysaccharide, a phosphorylated polysaccharide,    mannose, a phosphorylated mannose, a mannofuranose, a phosphorylated    mannofuranose, a mannopyranos, a phosphorylated mannopyranos,    mannose-1-phosphate, a nucleotide sugar, a uridine diphosphate, a    guanine diphosphate, a cytosine monophosphate, fucose, GDP-fucose, a    sialic acid, CMP-sialic acid, N-acetylneuraminic acid (Neu5Ac), and    CMP-Neu5Ac.-   87. The composition of embodiment 83 or 84, wherein the carbohydrate    is mannose-1-phosphate.-   88. The composition of embodiment 83 or 84, wherein the carbohydrate    is GDP-fucose.-   89. The composition of embodiment 83 or 84, wherein the carbohydrate    is CMP-sialic acid.-   90. The composition of any one of embodiments 83-89, wherein the    lipid particle comprises ethylene oxide, an ethylene oxide oligomer,    an ethylene oxide polymer, polyethylene glycol (PEG), or any    combination thereof.-   91. The composition of embodiment 90, wherein the lipid particle    comprises an ethylene oxide oligomer.-   92. The composition of embodiment 90, wherein the lipid particle    comprises an ethylene oxide polymer.-   93. The composition of embodiment 90, wherein the lipid particle    comprises polyethylene glycol (PEG).-   94. The composition of any one of embodiments 83-93, wherein the    lipid particle further comprises a lipid selected from the group    consisting of a phospholipid, a glycerolipid, a sphingolipid, and    any combination thereof; optionally wherein the lipid has a polar    head group.-   95. The composition of embodiment 94, wherein the polar head group    is selected from the group consisting of choline, ethanolamine,    glycerol, inositol, and any combination thereof.-   96. The composition of any one of embodiments 83-93, wherein the    lipid particle further comprises a phospholipid.-   97. The composition of embodiment 96, wherein the phospholipid is    selected from the group consisting of phosphatidylcholine (PC),    phosphatidylethanolamine (PE), phosphatidylglycerol (PG),    phosphatidylinositol (PI), and any combination thereof.-   98. The composition of any one of embodiments 83-97, wherein the    lipid particle further comprises a sterol.-   99. The composition of embodiment 98, wherein the sterol is    cholesterol.-   100. The composition of embodiment 99, wherein the cholesterol is    present at a concentration of at most 15 molar percent.-   101. The composition of any one of embodiments 83-100, wherein the    lipid particle further comprises a fatty acid.-   102. The composition of any one of embodiments 83-101, wherein the    lipid particle further comprises a PEGylated neutral lipid.-   103. The composition of any one of embodiments 83-102, wherein the    lipid particle is a lamellar nanostructure comprising an amphiphilic    lipid.-   104. The composition of any one of embodiments 83-103, wherein the    lipid particle is selected from the group consisting of a liposome,    a micelle, a solid lipid nanoparticle, and a niosome.-   105. The composition of any one of embodiments 83-103, wherein the    lipid particle is a liposome.-   106. The composition of embodiment 105, wherein the liposome    comprises polyethylene-glycol (PEG).-   107. The composition of embodiment 105 or 106, wherein the liposome    is a stealth liposome.-   108. The composition of embodiment 107, wherein the stealth liposome    is immunotolerant.-   109. The composition of any one of embodiments 105-108, wherein the    liposome further comprises a phospholipid selected from the group    consisting of phosphatidylcholine (PC), phosphatidylethanolamine    (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and any    combination thereof.-   110. The composition of any one of embodiments 105-108, wherein the    liposome further comprises phosphatidylcholine (PC) and    phosphatidylethanolamine (PE).-   111. The composition of any one of embodiments 105-110, wherein the    liposome comprises phosphatidylcholine (PC) at a concentration of at    least 70 molar percent.-   112. The composition of any one of embodiments 105-111, wherein the    liposome further comprises a PEGylated neutral lipid.-   113. The composition of embodiment 112, wherein the PEGylated    neutral lipid is present in the lipid particle at a concentration of    at least 1 molar percent.-   114. The composition of any one of embodiments 105-113, wherein the    liposome further comprises cholesterol.-   115. The composition of any one of embodiments 105-114, wherein the    liposome further comprises a fatty acid.-   116. The composition of embodiment 115, wherein the fatty acid    comprises a carbon chain that ranges in length from 14-18 carbon    atoms.-   117. The composition of any one of embodiments 83-103, wherein the    lipid particle is a micelle.-   118. The composition of any one of embodiments 83-103, wherein the    lipid particle is a solid lipid nanoparticle.-   119. The composition of any one of embodiments 83-103, wherein the    lipid particle is a niosome.-   120. The composition of any one of embodiments 83-119, wherein the    lipid particle has an average particle size that ranges from 0.05 to    0.5 microns in diameter.-   121. The composition of any one of embodiments 83-120, wherein the    lipid particle is capable of delivering the carbohydrate to a cell    interior.-   122. The composition of any one of embodiments 83-121, wherein the    composition when administered to a subject induces at least a    0.05-fold to at least a 3-fold increase in cellular production of    higher-order lipid-linked oligosaccharides in the subject, as    compared to cellular production of higher-order lipid-linked    oligosaccharides in the subject in the absence of the composition.-   123. The composition of embodiment 122, wherein the higher-order    lipid-linked oligosaccharides comprise Man4GlcNAc2, Man5GlcNAc2,    Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or any    combination thereof.-   124. A composition comprising:

a liposome; and

mannose-1-phosphate encapsulated in the liposome,

wherein the liposome comprises cholesterol and phosphatidylethanolamine(PE) attached to polyethylene glycol (PEG).

-   125. The composition of embodiment 124, wherein the liposome is a    stealth liposome.-   126. The composition of embodiment 125, wherein the stealth liposome    is immunotolerant.-   127. The composition of any one of embodiments 124-126, wherein the    liposome further comprises a phospholipid selected from the group    consisting of phosphatidylcholine (PC), phosphatidylserine (PS),    phosphatidylglycerol (PG), phosphatidylinositol (PI), and any    combination thereof.-   128. The composition of any one of embodiments 124-126, wherein the    liposome further comprises phosphatidylcholine (PC).-   129. The composition of embodiment 127 or 128, wherein the liposome    comprises phosphatidylcholine (PC) at a concentration of at least 70    molar percent.-   130. The composition of any one of embodiments 124-129, wherein the    liposome further comprises a PEGylated neutral lipid.-   131. The composition of embodiment 130, wherein the PEGylated    neutral lipid is present in the lipid particle at a concentration of    at least 1 molar percent.-   132. The composition of any one of embodiments 124-131, wherein the    cholesterol is present at a concentration of at most 15 molar    percent.-   133. The composition of any one of embodiments 124-132, wherein the    liposome further comprises a fatty acid.-   134. The composition of embodiment 133, wherein the fatty acid    comprises a carbon chain that ranges in length from 14-18 carbon    atoms.-   135. The composition of any one of embodiments 124-134, wherein the    liposome has an average particle size that ranges from 0.05 to 0.5    microns in diameter.-   136. The composition of any one of embodiments 124-135, wherein the    1 liposome is capable of delivering the mannose-1-phosphate to a    cell interior.-   137. The composition of any one of embodiments 124-136, wherein the    composition when administered to a subject induces at least a    0.05-fold to at least a 3-fold increase in cellular production of    higher-order lipid-linked oligosaccharides in the subject, as    compared to cellular production of higher-order lipid-linked    oligosaccharides in the subject in the absence of the composition.-   138. The composition of embodiment 137, wherein the higher-order    lipid-linked oligosaccharides comprise Man4GlcNAc2, Man5GlcNAc2,    Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or any    combination thereof.-   139. Use of the composition of any one of embodiments 1-138 in the    manufacture of a medicament for delivering a carbohydrate to a    subject.-   140. Use of the composition of any one of embodiments 1-138 in the    manufacture of a medicament for delivering a carbohydrate to a cell    interior.-   141. Use of the composition of any one of embodiments 1-138 in the    manufacture of a medicament for treating a congenital disorder of    glycosylation (CDG).-   142. The composition of any one of embodiments 1-138 for use in    delivering a carbohydrate to a subject in need thereof.-   143. The composition of any one of embodiments 1-138 for use in    delivering a carbohydrate to a cell interior of a subject in need    thereof.-   144. The composition of any one of embodiments 1-138 for use in    treating a congenital disorder of glycosylation (CDG) to a subject    in need thereof.-   145. A pharmaceutical composition comprising the composition of any    one of embodiments 1-138, and a pharmaceutically acceptable carrier.-   146. A kit comprising the composition of any one of embodiments    1-138.-   147. The kit of embodiment 146, wherein the kit further comprises a    package insert comprising instructions for using the composition to    treat a congenital disorder of glycosylation (CDG) in a subject in    need of such treatment.-   148. The kit of embodiment 146, wherein the kit further comprises a    package insert comprising instructions for using the composition to    deliver a carbohydrate to a subject in need of such treatment.-   149. The kit of embodiment 146, wherein the kit further comprises a    package insert comprising instructions for using the composition to    deliver a carbohydrate to a cell interior.-   150. A method for delivering a carbohydrate to a subject in need    thereof, comprising administering to the subject the composition of    any one of embodiments 1-138.-   151. The method of embodiment 150, wherein the subject has a    congenital disorder of glycosylation (CDG).-   152. The method of embodiment 151, wherein the congenital disorder    of glycosylation (CDG) is selected from the group consisting of a    CDG type I disorder, a CDG-Ia disorder, a CDG type II disorder, a    CDG-IIc disorder, and a CDG-IIf disorder.-   153. The method of embodiment 151, wherein the congenital disorder    of glycosylation (CDG) is a CDG-Ia disorder.-   154. A method for delivering a carbohydrate to a cell interior of a    subject in need thereof, comprising administering to the subject the    composition of any one of embodiments 1-138, wherein the    administered composition traverses the cell plasma membrane to    deliver the carbohydrate to the cell interior.-   155. The method of embodiment 154, wherein the subject has a    congenital disorder of glycosylation (CDG).-   156. The method of embodiment 155, wherein the congenital disorder    of glycosylation (CDG) is selected from the group consisting of a    CDG type I disorder, a CDG-Ia disorder, a CDG type II disorder, a    CDG-IIc disorder, and a CDG-IIf disorder.-   157. The method of embodiment 155, wherein the congenital disorder    of glycosylation (CDG) is a CDG-Ia disorder.-   158. A method for treating a congenital disorder of glycosylation    (CDG) in a subject in need thereof, comprising administering to the    subject the composition of any one of embodiments 1-138.-   159. The method of embodiment 158, wherein the congenital disorder    of glycosylation (CDG) is selected from the group consisting of a    CDG type I disorder, a CDG-Ia disorder, a CDG type II disorder, a    CDG-IIc disorder, and a CDG-IIf disorder.-   160. The method of embodiment 158, wherein the congenital disorder    of glycosylation (CDG) is a CDG-Ia disorder.-   161. The method of any one of embodiments 150-160, wherein    administration of the composition induces at least a 0.05-fold to at    least a 2-fold increase in cellular production of higher-order    lipid-linked oligosaccharides in the subject, as compared to    cellular production of higher-order lipid-linked oligosaccharides in    the subject in the absence of the composition.-   162. The method of embodiment 161, wherein the higher-order    lipid-linked oligosaccharides comprise Man4GlcNAc2, Man5GlcNAc2,    Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or any    combination thereof.-   163. The method of any one of embodiments 150-162, wherein    administration of the composition minimizes degradation of the lipid    particle and/or enhances retention of the lipid particle when    administered to the subject, and/or makes the lipid particle    immunotolerant when administered to the subject.-   164. The method of any one of embodiments 150-163, wherein the    composition is administered orally, topically, dermally, nasally,    intravenously, intramuscularly, intraperitoneally,    intracerobrospinally, intracranially, intraspinally, subcutaneously,    intra-articularly, intrasynovialy, or intrathecaly.-   165. Pharmaceutical nanocarriers of dolichol pyrophosphate linked    oligosaccharide to endoplasmic reticulum are capable of treating all    CDG type I disorders.-   166. Pharmaceutical nanocarriers of mannose-1 phosphate to cell    interior are capable of treating CDG-Ia disorder.-   167. Pharmaceutical nanocarriers of GDP-fucose to Golgi are capable    of treating CDG-IIc disorder.-   168. Pharmaceutical nanocarriers of CMP-sialic acid to Golgi are    capable of treating CDG-IIf disorder.

EXAMPLES

The invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure arehereby expressly incorporated by reference.

Abbreviations

“PC” corresponds to phosphatidylcholine.

“PE” corresponds to phosphatidylethanolamine.

“PEG” corresponds to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] or just Poly-ethylene-glycol.

“M1P” corresponds to mannose-1-phosphate.

“PBS” corresponds to phosphate buffered saline solution.

“MWCO” corresponds to molecular weight cut-off membrane.

Materials and Methods Liposomes

Materials for liposome preparation (PC, PE, and PE-PEG) were purchasedfrom Avanti Polar Lipids.

Cell Lines

CDG-Ia human dermal fibroblasts and wild-type human dermal fibroblastswere purchased from Coriell Institute.

Culture Media

Dulbecco's min. Eagle's media was purchased from Mediatech.

Alpha-minimal essential medium was purchased from Mediatech.

Glutamine and penicillin-streptomycin were purchased from OmegaScientific.

FBS was purchased from Thermo.

Example 1 Preparation of Mannose-1-Phosphate Liposomes

The following Example demonstrates the preparation of liposomescontaining mannose-1-phosphate (M1P-liposomes). The structure ofmannose-1-phosphate (M1P) is depicted in FIG. 1A. While a specificisomer is depicted in FIG. 1A, it should be understood that thecarbohydates used in the compositions and methods provided herein may beany isomeric form, provided that such carbohydrates are endogenouscarbohydrates for a given subject.

Liposome Preparation Method

Liposomes were prepared by thin-film hydration from phosphatidylcholine(PC), cholesterol, and phosphatidylethanolamine (PE) conjugatedtopoly-ethylene-glycol (PEG). The structure of PEG is depicted in FIG.1B. In FIG. 1B, in one variation, n may be in the range of 800-5000. Inother variations, the value of n may be 2000. In yet other variations,PEG may be modified either with meleimide or NHS-ester depending on whatthe PEG will be conjugated to. The structure of phosphatidylethanolamineattached to PEG (PEG-PE) is depicted in FIG. 1C. The structure ofphosphatidylcholine (PC) is depicted in FIG. 1D.

The bulk solvent from the lipid film of PC and cholesterol was removedby rotary evaporation for 30 minutes, followed by freeze drying for 4hours to remove trace amounts of solvent. Following this, the liposomeswere rehydrated first with a solution of mannose-1-phosphate (M1P) in 1×phosphate buffered saline pH 7.4 (PBS) at a M1P concentration of 10mg/mL. Then, PBS was further added so as to achieve a lipidconcentration of 20 mg/mL. Liposomes were then heated to 39° C. andsized or extruded through 200 nm pore size and then 100 nm pore sizepolycarbonate membranes using a mini handheld extruder. Liposomes werethen dialyzed against PBS using a 2 kDa molecular weight cut-offmembrane (MWCO).

Following this step,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (PEG) was added by post-insertion. Briefly, a thin film ofPEG was obtained by drying off solvent using a stream of N₂ gas andfreeze drying as mentioned previously. This film was then rehydratedusing the prepared M1P-loaded liposomal solution. After vortexing for 5minutes, the solution was incubated at 37° C. overnight to allow forpost-insertion of PEG into the core of the nanoparticle. The structureof the mannose-1-phosphate-loaded liposomes is depicted in FIG. 2.

TABLE 1 Molar Weight Formulation percentage (%) (mg) PC 70 16.7Cholesterol 30 3.64 DSPE-PEG₂₀₀₀ 2 1.75 M1P 5 1.0

In Table 1, the final volume of the liposome formulation was 1 mL.

Example 2 Measurement of M1P Content in Liposomes

CDG-Ia disorder is a gycosylation disorder characterized by thedeficiency or abnormalities associated with the enzymephosphomannomutase (PMM). This disorder is diagnosed by testing theserum glycoproteins of hepatic origin (transferrin is the clinicallytested protein), which are commonly underglycosylated. Typicallytransferrin is assessed using isoelectric focusing, which wouldunderscore a dramatic decrease in PMM activity (PMM activity in CDG-Iapatients is approximately 0.29 nM/min/mg, as compared to normal PMMactivity of approximately 2.97 nM/min/mg). PMM enzyme is responsible forcatalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate,which is in turn a precursor for the ultimate synthesis ofG₃M₉Gn₂-P-P-Dolichol, which in turn is a precursor tolipid-linked-oligosaccharide (LLO). This is represented by the followingschematic representation of the metabolic glycosylation pathway is:glucose→glucose-6-phosphate→fructose-6-phosphate→mannose→mannose-6-phosphate→mannose-1-phosphate→GDP-mannose→peptideN-glycosylation.

Typical LLO oligosaccharides include mannose subunits (M) linked to anN-acetylglucosamine disaccharide (Gn₂). The decrease in PMM activitythus hampers the full maturation of LLO oligosaccharides, whichtypically range in size from M₅Gn₂ to M₉Gn₂. The resulting immature LLOoligosaccharides are not fully functional LLO and are typicallytruncated. Immature LLO oligosaccharides typically have sizes of M₂Gn₂and M₃Gn₂.

Analysis of synthesized oligosaccharide chains is typically performed byincubating fibroblasts with radiolabeled mannose in order to verify anydivergence from established patterns of oligosaccharide maturation. Thisis typically accomplished by labeling newly-synthesized oligosaccharideswithin the human fibroblasts with [2-³H]mannose. Following incubationwith labeled mannose, cell lysates are digested to further analyze thelabeled sugars by chromatography. However, a steady supply of mannose isnecessary to ensure a continuous production of the saccharide chain. (S.Catherine Hubbard and Phillips W. Robbins. Journal of BiologicalChemistry (1980) 255: 11782-11793). A problem with the approach ofsystematically supplying mannose within the body to treat disorders,such as CDG-Ia, is the inability of mannose to traverse the plasmamembrane. Hence various mannose derivatives have been evaluated fortheir ability to accumulate within the cytosol of cells. However, thisapproach is also problematic, due to the toxicity and instability ofsuch derivatives.

The following Example demonstrates the capacity of M1P-liposomes todrive the biosynthesis and full maturation of various LLOoligosaccharides. This approach bypasses the need to either permeabilizecells or to use a mannose derivative.

Lipid Sample Preparation

Lipids were precipitated with 10% Triton X-100 using a ratio of 1:1liposome:detergent, respectively. Lipid precipitate was removed bycentrifugation. Isolated M1P was treated with HCl at a finalconcentration of 0.1 N; 1-phospho linkage was hydrolyzed for 15 min at100° C. Samples were dried down in a speed vacuum in preparation forlabeling with a fluorophore for detection and quantiation as describedby Alwael et al. (Anal. Methods, 2011, 3, 62). Concentration of M1P inliposomes was 1.0 mg per milliliter of formulation equivalent to 5% oftotal lipid concentration.

Therapeutic/Trials

Cellular internalization into the cytosol and subsequent integration ofthe M1P-liposomes (as prepared in Example 1), was assessed in vitrousing primary human dermal fibroblasts derived from patients affectedwith CDG-Ia disorder (CDG-Ia fibroblasts) and wild-type human dermalfibroblasts. For evaluation of the ability of the M1P-liposomes tocorrect protein glycosylation defects, M1P-liposomes were delivered toCDG Type I primary fibroblasts derived from patients affected withCDG-Ia disorder. The ability to drive glycosylation forward wasevaluated by comparing glycoprotein N-glycan structure before and aftertreatment. Such comparison required N-glycan analysis using normal phasechromatography.

By utilizing endocytotic pathways, M1P-liposomes can enter the cellinterior and release their cargo into the cytosol. Due to the ability todeliver a non-modified mannose-1-phosphate into the cytosol, analysiswas performed on the assembly of LLO oligosaccharides in wild-typefibroblasts and fibroblasts derived from a CDG-Ia patient (CDG-Iafibroblasts). LLO structures were isolated from the wild-typefibroblasts (having 100% PMM activity) as well as from the CDG-Iafibroblasts (having 10% PMM activity) and the amounts of the LLOstructures were compared using chromatographic methods.

CDG-Ia dermal fibroblasts make a series of truncated lipid-linkedoligosaccharide (LLO) species ranging in size from M₂Gn₂ to M₅Gn₂ withM₂Gn₂ and M₃Gn₂ being predominant. The M1P liposomes from Example 1 weretested in order to evaluate their capacity to normalize the distributionof various LLO oligosaccharides. CDG-Ia fibroblasts cultured in lowglucose DMEM media were incubated with 300 μM of bare M1P (M1P) and 300μM of the M1P-liposomes (M1PL). After treatment, cells were harvestedand LLO oligosaccharides were recovered, hydrolyzed, modified withfluorophore, and analyzed by normal phase chromatography as described byGao et al. (Methods in Enzymology, 2006, 415, 3-20) and Bones et al.(Anal. Chem, 2011, 83 (13), 5344-52). The peak area of each LLO wasnormalized to protein content prior to calculation of percentageabundance.

The results are depicted in Table 2. The distribution of various LLOoligosaccharides from wild-type fibroblasts ranged from M₂Gn₂ to M₇Gn₂.Treatment of CDG-Ia fibroblasts with M1P-liposomes inhibited thesynthesis of truncated LLO oligosaccharides M₂ Gn₂ and M₃Gn₂, andshifted production towards larger LLO oligosaccharide species causing anincrease in expression of M₅Gn₂-M₇Gn₂ structures (Table 2). The amountof M₅Gn₂ LLO produced after treatment of CDG-Ia fibroblasts withM1P-liposomes was three times greater than the amount in untreatedCDG-Ia fibroblasts, and twice the amount in CDG-Ia fibroblasts treatedwith bare M1P (Table 2). Additionally, higher order LLO oligosaccharides(e.g., M₆ Gn₂ and M₇Gn₂) were observed in CDG-Ia fibroblasts aftertreatment with M1P-liposomes (Table 2). Such higher order LLOoligosaccharides were not observed in the CDG-Ia fibroblasts prior totreatment with M1P-liposomes. Moreover, the amount of M₆Gn₂ and M₇Gn₂LLOoligosaccharides in CDG-Ia fibroblasts after treatment withM1P-liposomes was comparable to the amount in wild-type fibroblasts(Table 2).

Complete normalization was not observed in all cases, as the analyticalapproach that was utilized detected both newly synthesized LLOoligosaccharides and LLO oligosaccharides previously produced in thecell.

TABLE 2 Genotype Treatment M₂Gn₂ M₃Gn₂ M₄Gn₂ M₅Gn₂ M₆Gn₂ M₇Gn₂ NormalNone 18 10 36 32 4.5 0.4 CDG-Ia None 31 38 24 6.2 CDG-Ia 300 μM M1PL 1828 31 18 3.9 0.5 CDG-Ia 300 μM M1P 40 24 27 8.9

Table 2 depicts normal phase chromatography analyses of LLOoligosaccharide distribution (expressed as percentage, %) in wild-typefibroblasts and CDG-Ia fibroblasts. The LLO oligosaccharides contain twoor more mannose (Man) subunits and an N-acetylglucosamine disaccharide(GlcNAc2). In Table 2, “M₂Gn₂” corresponds to Man2GlcNAc2, “M₃Gn₂”corresponds to Man3GlcNAc2, “M₄Gn₂” corresponds to Man4GlcNAc2, “M₅Gn₂”corresponds to Man5GlcNAc2, “M₆Gnc₂” corresponds to Man6GlcNAc2, and“M₇Gn₂” corresponds to Man7GlcNAc2.

1. A composition comprising: a lipid particle; and an endogenouscarbohydrate encapsulated in the lipid particle, wherein the lipidparticle comprises a molecule capable of minimizing degradation of thelipid particle, enhancing retention of the lipid particle, and/or makingthe lipid particle immunotolerant when administered to a subject in needthereof.
 2. The composition of claim 1, wherein the molecule is ethyleneoxide, an ethylene oxide oligomer, an ethylene oxide polymer,polyethylene glycol (PEG), a PEGylated neutral lipid, or any combinationthereof.
 3. The composition of claim 2, wherein the lipid particlefurther comprises a lipid selected from the group consisting of aphospholipid, a glycerolipid, a sphingolipid, and any combinationthereof.
 4. The composition of claim 3, wherein the lipid has a polarhead group.
 5. The composition of claim 4, wherein the polar head groupcomprises choline, ethanolamine, serine, glycerol, inositol, or anycombination thereof.
 6. The composition of claim 1, wherein the lipidparticle further comprises phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylglycerol (PG), phosphatidylinositol (PI), or any combinationthereof.
 7. The composition of claim 1, wherein the lipid particlefurther comprises cholesterol, a fatty acid, or a combination thereof.8. The composition of claim 1, wherein the lipid particle is selectedfrom the group consisting of a liposome, a micelle, a solid lipidnanoparticle, and a niosome.
 9. The composition of claim 1, wherein theendogenous carbohydrate is selected from the group consisting of amonosaccharide, a phosphorylated monosaccharide, a disaccharide, aphosphorylated disaccharide, an oligosaccharide, a phosphorylatedoligosaccharide, a polysaccharide, a phosphorylated polysaccharide,mannose, a phosphorylated mannose, a mannofuranose, a phosphorylatedmannofuranose, a mannopyranos, a phosphorylated mannopyranos,mannose-1-phosphate, a nucleotide sugar, a uridine diphosphate, aguanine diphosphate, a cytosine monophosphate, fucose, GDP-fucose, asialic acid, CMP-sialic acid, N-acetylneuraminic acid (Neu5Ac), andCMP-Neu5Ac.
 10. The composition of claim 1, wherein the composition,when administered to a subject in need thereof, induces at least a0.05-fold to at least a 3-fold increase in cellular production ofhigher-order lipid-linked oligosaccharides in the subject, as comparedto cellular production of higher-order lipid-linked oligosaccharides inthe subject in the absence of administering the composition to thesubject.
 11. The composition of claim 10, wherein the higher-orderlipid-linked oligosaccharides comprise Man4GlcNAc2, Man5GlcNAc2,Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, or any combinationsthereof.
 12. A composition comprising: a liposome; andmannose-1-phosphate encapsulated in the liposome, wherein the liposomecomprises cholesterol and phosphatidylethanolamine (PE) attached topolyethylene glycol (PEG).
 13. A pharmaceutical composition comprisingthe composition of claim 1, and a pharmaceutically acceptable carrier.14. A method for delivering a carbohydrate to a subject in need thereof,comprising administering to the subject the composition of claim
 1. 15.A method for delivering a carbohydrate to a cell interior of a subjectin need thereof, comprising administering to the subject the compositionof claim 1, wherein the administered composition traverses the cellplasma membrane to deliver the carbohydrate to the cell interior.
 16. Amethod for treating a congenital disorder of glycosylation (CDG) in asubject in need thereof, comprising administering to the subject thecomposition of claim
 1. 17. The method of claim 16, wherein thecongenital disorder of glycosylation (CDG) is a CDG-Ia disorder.
 18. Themethod of claim 14, wherein administration of the composition induces atleast a 0.05-fold to at least a 2-fold increase in cellular productionof higher-order lipid-linked oligosaccharides in the subject, ascompared to cellular production of higher-order lipid-linkedoligosaccharides in the subject in the absence of administering thecomposition to the subject.
 19. The method of claim 18, wherein thehigher-order lipid-linked oligosaccharides comprise Man4GlcNAc2,Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, and any combination thereof. 20.The method of claim 15, wherein administration of the compositionminimizes degradation of the lipid particle, enhances retention of thelipid particle, and/or makes the lipid particle immunotolerant whenadministered to the subject.